Co-crystals and pharmaceutical compositions comprising the same

ABSTRACT

The present invention relates to compositions and co-crystals each comprising a compound of formula I having the structure: 
                         
wherein each of R 1  and R 2  is H or  2 H and a co-crystal former selected from adipic acid, citric acid, fumaric acid, maleic acid, succinic acid, or benzoic acid. Also within the scope of this invention are methods of making and using the same.

RELATED APPLICATIONS

This present application is a continuation of U.S. application Ser. No.15/130,266, filed Apr. 15, 2016, which is a continuation ofinternational PCT Application, PCT/US2014/061102, filed Oct. 17, 2014,which claims priority to U.S. Provisional Application No. 61/892,002filed on Oct. 17, 2013, the entire contents of which are incorporated byreference herein.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to co-crystals of DNA-dependent proteinkinase (DNA-PK) inhibitors. The invention also provides pharmaceuticalcompositions thereof and methods of using the co-crystals andcompositions in the treatment of cancer.

BACKGROUND OF THE INVENTION

Ionizing radiation (IR) induces a variety of DNA damage of which doublestrand breaks (DSBs) are the most cytotoxic. These DSBs can lead to celldeath via apoptosis and/or mitotic catastrophe if not rapidly andcompletely repaired. In addition to IR, certain chemotherapeutic agentsincluding topoisomerase II inhibitors, bleomycin, and doxorubicin alsocause DSBs. These DNA lesions trigger a complex set of signals throughthe DNA damage response network that function to repair the damaged DNAand maintain cell viability and genomic stability. In mammalian cells,the predominant repair pathway for DSBs is the Non-Homologous EndJoining Pathway (NHEJ). This pathway functions regardless of the phaseof the cell cycle and does not require a template to re-ligate thebroken DNA ends. NHEJ requires coordination of many proteins andsignaling pathways. The core NHEJ machinery consists of the Ku70/80heterodimer and the catalytic subunit of DNA-dependent protein kinase(DNA-PKcs), which together comprise the active DNA-PK enzyme complex.DNA-PKcs is a member of the phosphatidylinositol 3-kinase-related kinase(PIKK) family of serine/threonine protein kinases that also includesataxia telangiectasia mutated (ATM), ataxia telangiectasia andRad3-related (ATR), mTOR, and four PI3K isoforms. However, whileDNA-PKcs is in the same protein kinase family as ATM and ATR, theselatter kinases function to repair DNA damage through the HomologousRecombination (HR) pathway and are restricted to the S and G₂ phases ofthe cell cycle. While ATM is also recruited to sites of DSBs, ATR isrecruited to sites of single stranded DNA breaks.

NHEJ is thought to proceed through three key steps: recognition of theDSBs, DNA processing to remove non-ligatable ends or other forms ofdamage at the termini, and finally ligation of the DNA ends. Recognitionof the DSB is carried out by binding of the Ku heterodimer to the raggedDNA ends followed by recruitment of two molecules of DNA-PKcs toadjacent sides of the DSB; this serves to protect the broken terminiuntil additional processing enzymes are recruited. Recent data supportsthe hypothesis that DNA-PKcs phosphorylates the processing enzyme,Artemis, as well as itself to prepare the DNA ends for additionalprocessing. In some cases DNA polymerase may be required to synthesizenew ends prior to the ligation step. The auto-phosphorylation ofDNA-PKcs is believed to induce a conformational change that opens thecentral DNA binding cavity, releases DNA-PKcs from DNA, and facilitatesthe ultimate re-ligation of the DNA ends.

It has been known for some time that DNA-PK^(−/−) mice arehypersensitive to the effects of IR and that some non-selective smallmolecule inhibitors of DNA-PKcs can radiosensitize a variety of tumorcell types across a broad set of genetic backgrounds. While it isexpected that inhibition of DNA-PK will radiosensitize normal cells tosome extent, this has been observed to a lesser degree than with tumorcells likely due to the fact that tumor cells possess higher basallevels of endogenous replication stress and DNA damage (oncogene-inducedreplication stress) and DNA repair mechanisms are less efficient intumor cells. Most importantly, an improved therapeutic window withgreater sparing of normal tissue will be imparted from the combinationof a DNA-PK inhibitor with recent advances in precision delivery offocused IR, including image-guide RT (IGRT) and intensity-modulated RT(IMRT).

Inhibition of DNA-PK activity induces effects in both cycling andnon-cycling cells. This is highly significant since the majority ofcells in a solid tumor are not actively replicating at any given moment,which limits the efficacy of many agents targeting the cell cycle.Equally intriguing are recent reports that suggest a strong connectionbetween inhibition of the NHEJ pathway and the ability to killradioresistant cancer stem cells (CSCs). It has been shown in some tumorcells that DSBs in dormant CSCs predominantly activate DNA repairthrough the NHEJ pathway; it is believed that CSCs are usually in thequiescent phase of the cell cycle. This may explain why half of cancerpatients may experience local or distant tumor relapse despite treatmentas current strategies are not able to effectively target CSCs. A DNA-PKinhibitor may have the ability to sensitize these potential metastaticprogenitor cells to the effects of IR and select DSB-inducingchemotherapeutic agents.

Given the involvement of DNA-PK in DNA repair processes, DNA-PKinhibitory drugs may act as agents that enhance the efficacy of bothcancer chemotherapy and radiotherapy. The present invention featurescrystalline compositions of DNA-PK inhibitors together with a co-crystalformer (CCF), i.e., co-crystals. Compared to their free form(s), theco-crystals of the invention are advantageous as these compounds possessimproved dissolution, higher aqueous solubility, and greater solid statephysical stability than amorphous dispersions. The co-crystals describedherein also provide a reduced volume of the dosage form and thereforelower pill burden since these co-crystals also exhibit higher bulkdensities relative to amorphous forms. Further, the co-crystals of theinvention provide manufacturing advantages relative to amorphous formswhich require spray drying, lyophilization, or precipitation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an X-ray powder diffraction pattern of the co-crystalformed between Compound 1 with adipic acid.

FIG. 2 shows an X-ray powder diffraction pattern of the co-crystalformed between Compound 2 with adipic acid.

FIG. 3 shows an X-ray powder diffraction pattern of the co-crystalformed between Compound 1 with citric acid.

FIG. 4 shows an X-ray powder diffraction pattern of the co-crystalformed between Compound 1 and fumaric acid.

FIG. 5 shows an X-ray powder diffraction pattern of the co-crystalformed between Compound 1 and maleic acid.

FIG. 6 shows an X-ray powder diffraction pattern of the co-crystalformed between Compound 1 and succinic acid.

FIG. 7 shows an X-ray powder diffraction pattern of the co-crystalformed between Compound 1 and benzoic acid.

FIG. 8 shows a thermogravimetric analysis thermogram of the co-crystalformed between Compound 1 and adipic acid.

FIG. 9 shows a thermogravimetric analysis thermogram of the co-crystalformed between Compound 2 and adipic acid.

FIG. 10 shows a differential scanning calorimetry thermogram of theco-crystal formed between Compound 1 and adipic acid.

FIG. 11 shows a differential scanning calorimetry thermogram of theco-crystal formed between Compound 2 with adipic acid.

FIG. 12 shows a solid-state NMR spectrum of the co-crystal formedbetween Compound 1 and adipic acid.

FIG. 13 shows a solid-state NMR spectrum of the co-crystal formedbetween Compound 2 and adipic acid.

FIG. 14 shows an X-ray powder diffraction pattern of polymorphic Form Aof the co-crystal formed between Compound 1 with adipic acid.

FIG. 15 shows an X-ray powder diffraction pattern of polymorphic Form Bof the co-crystal formed between Compound 2 with adipic acid.

FIG. 16 shows a solid-state NMR spectrum of polymorphic Form A of theco-crystal formed between Compound 1 and adipic acid.

FIG. 17 shows a solid-state NMR spectrum of polymorphic Form A of theco-crystal formed between Compound 2 and adipic acid.

FIG. 18 shows a solid-state NMR spectrum of polymorphic Form B of theco-crystal formed between Compound 2 and adipic acid.

FIG. 19 shows a binary phase diagram of Compound 2 and adipic acid.

FIG. 20 shows a diagram of the calculated pH solubility of theco-crystal formed between Compound 2 with adipic acid (by excess adipicacid content) and free form Compound 2.

FIG. 21 shows two stage dissolution profiles for: i) Compound 1:adipicacid co-crystal prepared by hot melt extrusion and slurrycrystallization; ii) HME 65:35: Compound 1:adipic acid co-crystalmanufactured using hot melt extrusion with 65% w:w Compound 1 and 35%w:w adipic acid; iii) HME 75:25: Compound 1:adipic acid co-crystalmanufactured using hot melt extrusion with 75% w:w Compound 1 and 25%w:w adipic acid; iv) HME 80:20: Compound 1:adipic acid co-crystalmanufactured using hot melt extrusion with 80% w:w Compound 1 and 20%w:w adipic acid; v) SC 80:20: slurry crystallized Compound 2:adipic acidco-crystal with final Compound 2 content of 79% w:w Compound 2 and 21%w:w adipic acid; and vi) Free Form: Compound 2 free form.

FIG. 22 shows a predicted fraction absorbed for the co-crystal formedbetween Compound 2 and adipic acid, and Compound 2 free form.

FIG. 23 shows a diagram summarizing Bliss analysis of Compound (2) incombination with a panel of cytotoxic and non-cytotoxic agents.

FIG. 24 shows a diagram summarizing Bliss analysis of Compound (2) incombination with BMN-673 by tumor type.

FIG. 25 shows a diagram summarizing Bliss analysis of Compound (2) incombination with etoposide by tumor type.

FIG. 26 shows a diagram summarizing Bliss analysis of Compound (2) incombination with bleomycin by tumor type.

FIG. 27 shows a diagram summarizing Bliss analysis of Compound (2) incombination with erlotinib by tumor type.

FIG. 28 shows a diagram summarizing Bliss analysis of Compound (2) incombination with doxorubicin by tumor type.

FIG. 29 shows a diagram summarizing Bliss analysis of Compound (2) incombination with bleomycin by tumor type.

FIG. 30 shows a diagram summarizing Bliss analysis of Compound (2) inCombination with carboplatin by tumor type.

FIG. 31 shows a diagram summarizing Bliss analysis of Compound 1 orCompound 2 and standard of care combinations in primary human tumorchemosensitivity assays.

SUMMARY OF THE INVENTION

In a first aspect, the invention features a co-crystal comprising acompound of formula I

and a co-crystal former (CCF) selected from adipic acid, citric acid,fumaric acid, maleic acid, succinic acid, or benzoic acid, wherein eachof R¹ and R² is hydrogen or deuterium.

In another aspect, the invention provides a pharmaceutical compositionthat includes a co-crystal of a compound of formula I described above.In one embodiment, the pharmaceutical composition further includes adiluent, solvent, excipient, or carrier.

In yet another aspect, the invention provides a eutectic solidcomposition comprising: (a) a co-crystal comprising a compound offormula (I) and a co-crystal former selected from adipic acid, whereineach of R¹ and R² is hydrogen or deuterium, and wherein the molar ratioof the compound of formula I to adipic acid is about 2 to 1; and (b)adipic acid. In yet another aspect, the invention provides apharmaceutical composition comprising such a eutectic solid composition.In one embodiment, the pharmaceutical composition further includes adiluent, solvent, excipient, or carrier.

Another aspect of this invention provides a method of making aco-crystal of a compound of formula I and adipic acid, citric acid,fumaric acid, maleic acid, succinic acid, or benzoic acid. In oneembodiment, the method comprises: providing the compound of formula I;providing the co-crystal former; grinding, heating, co-subliming,co-melting, or contacting in solution the compound of formula I with theco-crystal former under crystallization conditions so as to form theco-crystal in solid phase; and then optionally isolating the co-crystalformed thereby. In another embodiment, the method comprises mixing acompound of formula (I) with adipic acid, citric acid, fumaric acid,maleic acid, succinic acid, or benzoic acid at an elevated temperatureto form the co-crystal. In some embodiments, the making a co-crystal ofa compound of formula I and the CCF includes providing the compound offormula I and adipic acid, citric acid, fumaric acid, maleic acid,succinic acid, or benzoic acid in a molar ratio between about 1 to 1.2to about 1 to 3.6, respectively.

In yet another aspect, the invention provides a method for modulating achemical or physical property of interest (such as melting point,solubility, dissolution, hygroscopicity, and bioavailability) of aco-crystal containing a compound of formula I and adipic acid, citricacid, fumaric acid, maleic acid, succinic acid, or benzoic acid. Themethod includes the steps of measuring the chemical or physical propertyof interest for the compound of formula I and CCF; determining the molefraction of the compound of formula I and CCF that will result in thedesired modulation of the chemical or physical property of interest; andpreparing the co-crystal with the molar fraction as determined.

The compositions and co-crystals of this invention can be used fortreating diseases implicated by or associated with the inhibition ofDNA-PK. In particular, the invention provides a method of sensitizing acell to an agent that induces a DNA lesion comprising contacting thecell with a co-crystal of the invention or pharmaceutical compositionthereof.

The invention further provides methods of potentiating a therapeuticregimen for treatment of cancer comprising administering to anindividual in need thereof an effective amount of a co-crystal of theinvention or pharmaceutical composition thereof. In one embodiment, thetherapeutic regimen for treatment of cancer includes radiation therapy.

The present invention also provides methods of treating cancer in ananimal that includes administering to the animal an effective amount ofa co-crystal or pharmaceutical composition of the invention. Theinvention further is directed to methods of inhibiting cancer cellgrowth, including processes of cellular proliferation, invasiveness, andmetastasis in biological systems. Methods include use of such aco-crystal or pharmaceutical composition to inhibit cancer cell growth.

The invention provides a method of inhibiting DNA-PK activity in abiological sample that includes contacting the biological sample with aco-crystal or pharmaceutical composition of the invention.

Also within the scope of this invention is a method of treating diseasesdescribed herein, such as cancer, which comprising administering to asubject in need thereof a therapeutically effective amount of aco-crystal of this invention or a composition of this invention.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention is directed to co-crystals comprising acompound of formula I

and a co-crystal former (CCF) selected from adipic acid, citric acid,fumaric acid, maleic acid, succinic acid, or benzoic acid, wherein eachof R¹ and R² is hydrogen or deuterium.

In one embodiment, the compound of formula I is(S)—N-methyl-8-(1-((2′-methyl-[4,5′-bipyrimidin]-6-yl)amino)propan-2-yl)quinoline-4-carboxamide(Compound 1).

In another embodiment, the compound of formula I is(S)—N-methyl-8-(1-((2′-methyl-4′,6′-dideutero-[4,5′-bipyrimidin]-6-yl)amino)propan-2-yl)quinoline-4-carboxamide(Compound 2).

In one embodiment, the invention provides a co-crystal that includes acompound of formula I and adipic acid as the CCF. In a furtherembodiment, the X-ray powder diffraction (XRPD) pattern of thisco-crystal exhibits peaks at about 6.46, 7.91, 11.92, 12.26, 12.99,14.19, 18.68, and 19.07-Theta. In another embodiment, the XRPD patternof this co-crystal exhibits peaks as shown in FIG. 1. In yet anotherembodiment, the XRPD pattern of this co-crystal exhibits peaks as shownin FIG. 2. In yet another further embodiment, its differential scanningcalorimetry (DSC) thermogram shows melting points at about 195° C. andabout 245° C.

In one embodiment, the invention provides a co-crystal that includes acompound of formula I and citric acid as the CCF. In one embodiment, theXRPD pattern of this co-crystal exhibits peaks at about 7.44, 8.29,11.35, 13.26, 15.49, 21.55, and 23.57-Theta. In another embodiment, theXRPD pattern of this co-crystal exhibits peaks as shown in FIG. 3. Inyet another embodiment, a compound of formula I and the CCF are both inthe solid state (e.g., crystalline) and are bonded non-covalently (i.e.,by hydrogen bonding).

In one embodiment, the invention provides a co-crystal that includes acompound of formula I and fumaric acid as the CCF. In one embodiment,the XRPD pattern of this co-crystal exhibits peaks at about 8.26, 10.11,14.97, 16.61, 17.22, 25.20, and 26.01-Theta. In another embodiment, theXRPD pattern of this co-crystal exhibits peaks as shown in FIG. 4. Inyet another embodiment, a compound of formula I and the CCF are both inthe solid state (e.g., crystalline) and are bonded non-covalently (i.e.,by hydrogen bonding).

In one embodiment, the invention provides a co-crystal that includes acompound of formula I and maleic acid as the CCF. In one embodiment, theXRPD pattern of this co-crystal exhibits peaks at about 6.21, 10.43,11.28, 12.41, 13.26, 18.87, and 21.08-Theta. In another embodiment, theXRPD pattern of this co-crystal exhibits peaks as shown in FIG. 5. Inyet another embodiment, a compound of formula I and the CCF are both inthe solid state (e.g., crystalline) and are bonded non-covalently (i.e.,by hydrogen bonding).

In one embodiment, the invention provides a co-crystal that includes acompound of formula I and succinic acid as the CCF. In one embodiment,the XRPD pattern of this co-crystal exhibits peaks at about 8.02, 12.34,14.78, 17.32, 19.56, and 20.06-Theta. In another embodiment, the XRPDpattern of this co-crystal exhibits peaks as shown in FIG. 6. In anotherembodiment, a compound of formula I and the CCF are both in the solidstate (e.g., crystalline) and are bonded non-covalently (i.e., byhydrogen bonding).

In yet another embodiment, the invention provides a co-crystal thatincludes a compound of formula I and benzoic acid as the CCF. In oneembodiment, the XRPD pattern of this co-crystal exhibits peaks at 8.70,13.90, 15.62, 17.65, 18.15, 20.77, and 24.72-Theta. In anotherembodiment, the XRPD pattern of this co-crystal exhibits peaks as shownin FIG. 7. In another embodiment, a compound of formula I and the CCFare both in the solid state (e.g., crystalline) and are bondednon-covalently).

In one embodiment, the invention provides co-crystals of the formula(Compound 1)_(n):(AA)_(m), wherein n is 1 and m is between 0.4 and 2.1.In one embodiment, n is 1 and m is between 0.9 and 3.1. In oneembodiment for co-crystals comprising adipic acid, n is about 2 and m isabout 1. In one embodiment for co-crystals comprising adipic acid, n isabout 2 and m is about 1.

In another embodiment, the invention provides co-crystals of the formula(Compound 2)_(n):(AA)_(m), wherein n is 1 and m is between 0.4 and 2.1In one embodiment for co-crystals comprising adipic acid, n is about 2and m is about 1.

In another embodiment, the invention provides a co-crystal of a compoundof formula I and CCF adipic acid, citric acid, fumaric acid, maleicacid, succinic acid, or benzoic acid, wherein the co-crystal is a solidat the room temperature and the compound of formula I and CCF interactby noncovalent bonds. In certain embodiments, the non-covalent bondinteractions between the compound of formula I and CCF include hydrogenbonding and van der Waals interactions. In one embodiment, the CCF isadipic acid.

In one embodiment, the invention provides a co-crystal of Compound (1)and CCF adipic acid, wherein the molar ratio of Compound (1) to adipicacid is about 2:1.

In another embodiment, the invention provides a co-crystal of Compound(2) and CCF adipic acid, wherein the molar ratio of Compound (2) toadipic acid is about 2:1.

In another embodiment, the co-crystal of Compound (2) and CCF adipicacid (adipic acid co-crystal of Compound (2)) is in polymorphic Form Aor B. Polymorphic Forms A and B are two conformational polymorphs of theadipic acid co-crystal of Compound (2). In yet another embodiment, theco-crystal of Compound (1) and CCF adipic acid (adipic acid co-crystalof Compound (1)) is in polymorphic Form A or B. Polymorphic Forms A andB are two conformational polymorphs of the adipic acid co-crystal ofCompound (1), and their ¹³C solid state nuclear magnetic resonancespectroscopies are essentially the same as those for Polymorphic Forms Aand B of Compound (2).

In a specific embodiment, the polymorphic Form A is characterized by ¹³Csolid state nuclear magnetic resonance spectroscopy peaks at about117.1, 96.8, 95.7, 27.6, 14.8 ppm. In another specific embodiment, thepolymorphic Form A is characterized by ¹³C solid state nuclear magneticresonance spectroscopy peaks at about 161.6, 154.5, 117.1, 96.8, 95.7,51.5, 50.2, 27.6, 25.6, 18.5, and 14.8 ppm. In yet another specificembodiment, the polymorphic Form A is characterized by ¹³C solid statenuclear magnetic resonance spectroscopy peaks at about 179.4, 168.4,161.6, 158.3, 154.5, 147.8, 145.7, 143.2, 141.8, 124.6, 117.1, 96.8,95.7, 51.5, 50.2, 31.2, 30.1, 27.6, 25.6, 18.5, and 14.8 ppm. In yetanother specific embodiment, the polymorphic Form A is characterized by¹³C solid state nuclear magnetic resonance spectroscopy peaks as shownin FIG. 16 or 17.

In a specific embodiment, the polymorphic Form B is characterized by ¹³Csolid state nuclear magnetic resonance spectroscopy peaks at about117.9, 97.3, 94.0, 26.7, and 15.7 ppm. In another specific embodiment,the polymorphic Form B is characterized by ¹³C solid state nuclearmagnetic resonance spectroscopy peaks at about 161.7, 153.8, 117.9,97.3, 94.0, 50.7, 25.3, 26.7, 18.8, and 15.7 ppm. In yet anotherspecific embodiment, the polymorphic Form B is characterized by ¹³Csolid state nuclear magnetic resonance spectroscopy peaks at about179.1, 168.3, 158.1, 147.2, 142.4, 125.8, 124.5, 117.9, 97.3, 94.0,32.3, 30.1, 26.7, and 15.7 ppm. In yet another specific embodiment, thepolymorphic Form B is characterized by ¹³C solid state nuclear magneticresonance spectroscopy peaks as shown in FIG. 17.

In yet another embodiment, the co-crystal of Compound (2) and CCF adipicacid (adipic acid co-crystal of Compound (2)) is in a mixture ofpolymorphic Forms A and B. In yet another embodiment, the co-crystal ofCompound (1) and CCF adipic acid (adipic acid co-crystal of Compound(1)) is in a mixture of polymorphic Forms A and B.

The present invention encompasses the co-crystals of a compound offormula I and CCF described above in isolated, pure form, or in amixture as a solid composition when admixed with other materials, forexample, free form of compound of formula I or free CCF. In oneembodiment, the invention provides pharmaceutically acceptablecompositions comprising the co-crystals of a compound of formula I andthe CCF described above and an additional free CCF. In a specificembodiment, the compositions comprise the co-crystals of Compound (1) or(2) and CCF adipic acid described above and additional adipic acid. Insome specific embodiments, the overall molar ratio of the compound offormula I to CCF (both part of the co-crystals and free CCF, e.g., adpicacid in the co-crystals and free adipic acid) in such compositions is ina range from about 1:0.55 to about 1:100. In other specific embodiments,the overall molar ratio of the compound of formula I to CCF in suchcompositions is in a range from about 1:0.55 to about 1:50. In otherspecific embodiments, the overall molar ratio of the compound of formulaI to CCF in such compositions is in a range from about 1:0.55 to about1:10. In some specific embodiments, the overall weight ratio of thecompound of formula I to CCF in such compositions is in a range fromabout 85 wt %:15 wt % to about 60 wt %:40 wt %. In other specificembodiments, the overall weight ratio of the compound of formula I toCCF is in a range from about 70 wt %:30 wt % to about 60 wt %:40 wt %.In yet other embodiments, the overall weight ratio of the compound offormula I to CCF is about 65 wt %:35 wt %.

In another embodiment, the invention provides eutectic solidcompositions comprising: (a) a co-crystal comprising a compound offormula (I), and a CCF selected from adipic acid, wherein each of R¹ andR² is hydrogen or deuterium, and wherein the molar ratio of the compoundof formula I to adipic acid is about 2 to 1; and (b) adipic acid. Asused herein, the term “eutectic solid” means a solid material resultingfrom a eutectic reaction known in the art. Without being bound to aparticular theory, an eutectic reaction is defined as follows:

In the eutection reaction, a single liquid phase and two solid phasesall co-exist at the same time and are in chemical equilibrium. It formsa super-lattice or microstructure on cooling which releases at once allits components into a liquid mixture (melts) at a specific temperature(the eutectic temperature).

In one embodiment, the overall weight ratio of the compound of formula Ito adipic acid in the eutectic solid compositions is in a range fromabout 70 wt %:30 wt % to about 60 wt %:40 wt %. In yet anotherembodiment, the overall weight ratio of the compound of formula I toadipic acid is in a range from about 65 wt %:35 wt %. In yet anotherembodiment, the molar ratio of the co-crystal of a compound of formula Ito adipic acid is about 1 to 1.03.

The pure form means that the particular co-crystal or polymorphic formcomprises over 95% (w/w), for example, over 98% (w/w), over 99% (w/w %),over 99.5% (w/w), or over 99.9% (w/w).

More specifically, the present invention also provides pharmaceuticallyacceptable compositions where each of the co-crystals or polymorphicforms are in the form of a composition or a mixture of the polymorphicform with one or more other crystalline, solvate, amorphous, or otherpolymorphic forms or their combinations thereof. For example, in oneembodiment, the compositions comprise Form A of the adipic acidco-crystal of Compound (2) along with one or more other polymorphicforms of Compound (2), such as amorphous form, hydrates, solvates,and/or other forms or their combinations thereof. In a specificembodiment, the compositions comprise Form A of the adipic acidco-crystal of Compound (2) along with Form B of the adipic acidco-crystal of Compound (2). More specifically, the composition maycomprise from trace amounts up to 100% of the specific polymorphic formor any amount, for example, in a range of 0.1%-0.5%, 0.1%-1%, 0.1%-2%,0.1%-5%, 0.1%-10%, 0.1%-20%, 0.1%-30%, 0.1%-40%, 0.1%-50%, 1%-50%, or10%-50% by weight based on the total amount of the compound of formula Iin the composition. Alternatively, the composition may comprise at least50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5% or 99.9% by weight ofspecific polymorphic form based on the total amount of the compound offormula I in the composition.

In one embodiment, the compounds in accordance with the presentinvention are provided in the form of a single enantiomer at least 95%,at least 97% and at least 99% free of the corresponding enantiomer.

In a further embodiment, the compounds in accordance with the presentinvention are in the form of the (+) enantiomer at least 95% free of thecorresponding (−) enantiomer.

In a further embodiment, the compounds in accordance with the presentinvention are in the form of the (+) enantiomer at least 97% free of thecorresponding (−) enantiomer.

In a further embodiment, the compounds in accordance with the presentinvention are in the form of the (+) enantiomer at least 99% free of thecorresponding (−) enantiomer.

In a further embodiment, the compounds in accordance with the presentinvention are in the form of the (−) enantiomer at least 95% free of thecorresponding (+) enantiomer.

In a further embodiment, the compounds in accordance with the presentinvention are in the form of the (−) enantiomer at least 97% free of thecorresponding (+) enantiomer.

In a further embodiment the compounds in accordance with the presentinvention are in the form of the (−) enantiomer at least 99% free of thecorresponding (+) enantiomer.

The present invention also provides methods of making the co-crystalsdescribed above. In one embodiment, the methods comprises grinding,heating, co-subliming, co-melting, or contacting either(S)—N-methyl-8-(1-((2′-methyl-[4,5′-bipyrimidin]-6-yl)amino)propan-2-yl)quinoline-4-carboxamideor(S)—N-methyl-8-(1-((2′-methyl-4′,6′-dideutero-[4,5′-bipyrimidin]-6-yl)amino)propan-2-yl)quinoline-4-carboxamidewith the co-crystal former under crystallization conditions so as toform the co-crystal in solid phase, wherein the co-crystal former isselected from adipic acid, citric acid, fumaric acid, maleic acid,succinic acid, or benzoic acid.

In another embodiment, the methods comprises mixing a compound offormula (I) with a CCF selected from adipic acid, citric acid, fumaricacid, maleic acid, succinic acid, or benzoic acid at an elevatedtemperature to form the co-crystal. The compound of formula (I) can bemixed with the CCF to generate a mixture of the compound and CCF, andthen the mixture of the compound and CCF are heated at an elevatedtemperature to form the co-crystal. Alternatively, the mixing andheating steps can be performed at the same time.

In one specific embodiment, the CCF is adipic acid, and the compound offormula (I) is mixed with adipic acid at an elevated temperature in arange of about 110° C. and about 195° C. to form the co-crystal. Inanother specific embodiment, the elevated temperature is in a range ofabout 130° C. and about 180° C., or in a range of about 140° C. andabout 160° C.

In another specific embodiment, the CCF is adipic acid, and 10 wt % toabout 85 wt % of the compound (I) and about 90 wt % to 15 wt % of adipicacid are mixed. In yet another specific embodiment, about 30 wt % toabout 80 wt % and the adipic acid is about 70 wt % to about 20 wt %. Inyet another specific embodiment, the compound (I) is about 50 wt % toabout 80 wt % and the adipic acid is about 50 wt % to about 20 wt %. Inyet another specific embodiment, the compound (I) is about 60 wt % to 70wt % and the adipic acid is about 40 wt % to about 30 wt %. In yetanother specific embodiment, the compound (I) is about 65 wt % and theadipic acid is about 35 wt %.

In yet another embodiment, the methods include: providing the compoundof formula I; providing the co-crystal former; grinding, heating,co-subliming, co-melting, or contacting in solution the compound offormula I with the co-crystal former under crystallization conditions soas to form the co-crystal in solid phase; and then optionally isolatingthe co-crystal formed thereby. In some specific embodiments, the makinga co-crystal of a compound of formula I and the CCF includes providingthe compound of formula I and adipic acid, citric acid, fumaric acid,maleic acid, succinic acid, or benzoic acid in a molar ratio betweenabout 1 to 0.55 to about 1 to 3.6, respectively. In some specificembodiments, the making a co-crystal of a compound of formula I and theCCF includes providing the compound of formula I and adipic acid, citricacid, fumaric acid, maleic acid, succinic acid, or benzoic acid in amolar ratio between about 1 to 1.2 to about 1 to 3.6, respectively.

In yet another embodiment, the invention provides methods for modulatinga chemical or physical property of interest (such as melting point,solubility, dissolution, hygroscopicity, and bioavailability) of aco-crystal containing a compound of formula I and adipic acid, citricacid, fumaric acid, maleic acid, succinic acid, or benzoic acid. Themethods include: measuring the chemical or physical property of interestfor the compound of formula I and CCF; determining the mole fraction ofthe compound of formula I and CCF that will result in the desiredmodulation of the chemical or physical property of interest; andpreparing the co-crystal with the molar fraction as determined.

As used herein, the following definitions shall apply unless otherwiseindicated. For purposes of this invention, the chemical elements areidentified in accordance with the Periodic Table of the Elements, CASversion, and the Handbook of Chemistry and Physics, 75^(th) Ed. 1994.Additionally, general principles of organic chemistry are described in“Organic Chemistry,” Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry,” 5^(th) Ed.,Smith, M. B. and March, J., eds. John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference/

For XRPD peak assignments, the term “about” means a range of +/−0.2relative to the stated value. For ¹³C solid state NMR spectra, the term“about” means a range of +/−0.1 relative to the stated value. Otherwise,the term “about” means a value of +/−10% of the stated value. When thisterm is followed by a series of numbers it applies to each of thenumbers in the series.

For compounds of the invention in which R¹ or R² is deuterium, thedeuterium to hydrogen ratio is at least 5 to 1. In some embodiments, thedeuterium to hydrogen ratio is at least 9 to 1. In other embodiments,the deuterium to hydrogen ratio is at least 19 to 1.

Methods for preparing and characterizing a co-crystal are welldocumented in the literature. See, e.g., Trask et al., Chem. Commun.,2004, 890-891; and 0. Almarsson and M. J. Zaworotko, Chem. Commun.,2004, 1889-1896. These methods in general are also suitable forpreparing and characterizing co-crystals of this invention.

Examples of preparing co-crystals with an active pharmaceuticalingredient and a CCF include hot-melt extrusion, ball-milling, meltingin a reaction block, evaporating solvent, slurry conversion, blending,sublimation, or modeling. In the ball-milling method, certain molarratios of the components of the co-crystal (e.g., a compound ofinterest, such as a compound of formula I of this invention, and a CCF)are mixed and milled with balls. Optionally, a solvent such as methylethyl ketone, chloroform, and/or water can be added to the mixture beingball milled. After milling, the mixture can be dried under vacuum eitherat the room temperature or in the heated condition, which typicallygives a powder product. In the melting method, the components of aco-crystal (e.g., a CCF and a compound of formula I) are mixed,optionally with a solvent such as acetonitrile. The mixture is thenplaced in a reaction block with the lid closed, and then heated to theendotherm. The resulting mixture is then cooled off and solvent, ifused, removed. In the solvent-evaporation method, each component of aco-crystal is first dissolved in a solvent (e.g., a solvent mixture,such as methanol/dichloromethane azeotrope, or toluene/acetonitrile(e.g., 50/50 by volume)), and the solutions are then mixed together. Themixture is then allowed to sit and solvent to evaporate to dryness, toyield the co-crystal. In the hot-melt extrusion (HME) method, a newmaterial (the extrudate) is formed by forcing it through an orifice ordie (extruder) under controlled conditions, such as temperature, mixing,feed-rate and pressure. An extruder typically comprises a platform thatsupports a drive system, an extrusion barrel, a rotating screw arrangedon a screw shaft and an extrusion die for defining product shape.Alternatively, the extrusion die can be removed and the product can beshaped by other means. Typically, process parameters are controlled viaconnection to a central electronic control unit. The extrusion drivesystem generally comprises motor, gearbox, linkage and thrust bearings,whereas the barrel and screw is commonly utilized in a modularconfiguration. Any suitable HME technologies known in the art, forexample, Gavin P. Andrews et al., “Hot-melt extrusion: an emerging drugdelivery technology”, Pharmaceutical Technology Europe, volume 21, Issue1 (2009), can be used in the invention. In one embodiment, theco-crystals of the invention are prepared by hot-melt extrusion.

Examples of characterization methods include thermogravimetric analysis(TGA), differential scanning calorimetry (DSC), X-ray powder diffraction(XRPD), solid-state nuclear magnetic resonance spectroscopy (ss-NMR),solubility analyses, dynamic vapor sorption, infrared off-gas analysis,and suspension stability. TGA can be used to investigate the presence ofresidual solvents in a co-crystal sample and to identify the temperatureat which decomposition of each co-crystal sample occurs. DSC can be usedto look for thermotransitions occurring in a co-crystal sample as afunction of temperature and determine the melting point of eachco-crystal sample. XRPD can be used for structural characterization ofthe co-crystal. Solubility analysis can be performed to reflect thechanges in the physical state of each co-crystal sample. Suspensionstability analysis can be used to determine the chemical stability of aco-crystal sample in a solvent.

Pharmaceutically Acceptable Salts

The present invention also covers co-crystals formed withpharmaceutically acceptable salts of the compounds of formula I. Also,the combination therapy of the invention discussed below includesadministering the compounds of formula I and pharmaceutically acceptablesalts thereof, and their co-crystals described herein. The compounds offormula I can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable salt.

A “pharmaceutically acceptable salt” means any non-toxic salt of acompound of this invention that, upon administration to a recipient, iscapable of providing, either directly or indirectly, a compound of thisinvention or an inhibitorily active metabolite or residue thereof. Asused herein, the term “inhibitorily active metabolite or residuethereof” means that a metabolite or residue thereof is also a DNA-PKinhibitor.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge et al., describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporatedherein by reference. Pharmaceutically acceptable salts of the compoundsof this invention include those derived from suitable inorganic andorganic acids and bases. These salts can be prepared in situ during thefinal isolation and purification of the compounds. Acid addition saltscan be prepared by 1) reacting the purified compound in its free-basedform with a suitable organic or inorganic acid and 2) isolating the saltthus formed.

Examples of pharmaceutically acceptable, nontoxic acid addition saltsare salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchloric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, glycolate, gluconate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate,lauryl sulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like.

Base addition salts can be prepared by 1) reacting the purified compoundin its acid form with a suitable organic or inorganic base and 2)isolating the salt thus formed. Salts derived from appropriate basesinclude alkali metal (e.g., sodium, lithium, and potassium), alkalineearth metal (e.g., magnesium and calcium), ammonium and N⁺(C₁₋₄alkyl)₄salts. This invention also envisions the quaternization of any basicnitrogen-containing groups of the compounds disclosed herein. Water oroil-soluble or dispersible products may be obtained by suchquaternization.

Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, lower alkyl sulfonate and aryl sulfonate. Other acids andbases, while not in themselves pharmaceutically acceptable, may beemployed in the preparation of salts useful as intermediates inobtaining the compounds of the invention and their pharmaceuticallyacceptable acid or base addition salts.

Uses of the Co-Crystals and Pharmaceutical Compositions of the Invention

An effective amount of a co-crystal or pharmaceutical composition of theinvention can be used to treat diseases implicated or associated withthe cancer. An effective amount is the amount which is required toconfer a therapeutic effect on the treated subject, e.g. a patient. Asused herein, the terms “subject” and “patient” are used interchangeably.The terms “subject” and “patient” refer to an animal (e.g., a bird suchas a chicken, quail or turkey, or a mammal), specifically a “mammal”including a non-primate (e.g., a cow, pig, horse, sheep, rabbit, guineapig, rat, cat, dog, and mouse) and a primate (e.g., a monkey, chimpanzeeand a human), and more specifically a human. In one embodiment, thesubject is a non-human animal such as a farm animal (e.g., a horse, cow,pig or sheep), or a pet (e.g., a dog, cat, guinea pig or rabbit). In apreferred embodiment, the subject is a “human”.

The precise amount of compound administered to a subject will depend onthe mode of administration, the type and severity of the cancer and onthe characteristics of the subject, such as general health, age, sex,body weight and tolerance to drugs. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors. Whenco-administered with other agents, e.g., when co-administered with ananti-cancer medication, an “effective amount” of the second agent willdepend on the type of drug used. Suitable dosages are known for approvedagents and can be adjusted by the skilled artisan according to thecondition of the subject, the type of condition(s) being treated and theamount of a compound described herein being used. In cases where noamount is expressly noted, an effective amount should be assumed.Generally, dosage regimens can be selected in accordance with a varietyof factors including the disorder being treated and the severity of thedisorder; the activity of the specific compound employed; the specificcomposition employed; the age, body weight, general health, sex and dietof the patient; the time of administration, route of administration, andrate of excretion of the specific compound employed; the renal andhepatic function of the subject; and the particular compound or saltthereof employed, the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The skilled artisan canreadily determine and prescribe the effective amount of the compoundsdescribed herein required to treat, to prevent, inhibit (fully orpartially) or arrest the progress of the disease.

The effective amount of a co-crystal or pharmaceutical composition ofthe invention is between about 0.1 to about 200 mg/kg body weight/day.In one embodiment, the effective amount of a co-crystal orpharmaceutical composition of the invention is between about 1 to about50 mg/kg body weight/day. In another embodiment, the effective amount ofa co-crystal or pharmaceutical composition of the invention is betweenabout 2 to about 20 mg/kg body weight/day. Effective doses will alsovary, as recognized by those skilled in the art, dependent on route ofadministration, excipient usage, and the possibility of co-usage withother therapeutic treatments including use of other therapeutic agentsand/or therapy.

The co-crystals or pharmaceutical compositions of the invention can beadministered to the subject in need thereof (e.g., cells, a tissue, or apatient (including an animal or a human) by any method that permits thedelivery of a compound of formula I, e.g., orally, intravenously, orparenterally. For instance, they can be administered via pills, tablets,capsules, aerosols, suppositories, liquid formulations for ingestion orinjection.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. In Remington: TheScience and Practice of Pharmacy, 21st edition, 2005, ed. D. B. Troy,Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia ofPharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, New York, the contents of each of which isincorporated by reference herein, are disclosed various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention.

A pharmaceutically acceptable carrier may contain inert ingredientswhich do not unduly inhibit the biological activity of the compounds.The pharmaceutically acceptable carriers should be biocompatible, e.g.,non-toxic, non-inflammatory, non-immunogenic or devoid of otherundesired reactions or side-effects upon the administration to asubject. Standard pharmaceutical formulation techniques can be employed.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, ion exchangers,alumina, aluminum stearate, lecithin, serum proteins (such as humanserum albumin), buffer substances (such as twin 80, phosphates, glycine,sorbic acid, or potassium sorbate), partial glyceride mixtures ofsaturated vegetable fatty acids, water, salts or electrolytes (such asprotamine sulfate, disodium hydrogen phosphate, potassium hydrogenphosphate, sodium chloride, or zinc salts), colloidal silica, magnesiumtrisilicate, polyvinyl pyrrolidone, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, methylcellulose,hydroxypropyl methylcellulose, wool fat, sugars such as lactose, glucoseand sucrose; starches such as corn starch and potato starch; celluloseand its derivatives such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients such as cocoa butter and suppository waxes; oils suchas peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil;corn oil and soybean oil; glycols; such a propylene glycol orpolyethylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

In one specific example, the pharmaceutically acceptable compositions ofthe invention comprise methylcellulose, such as about 0.5 wt %methylcellulose. In another specific example, the pharmaceuticallyacceptable compositions of the invention comprise methylcellulose andbenzoic acid, such as about 0.5 wt % methylcellulose and about 0.2 wt %benzoic acid. In another specific example, the pharmaceuticallyacceptable compositions comprise methylcellulose and benzoic acid, suchas about 0.5 wt % methylcellulose, about 0.1 wt % benzoic acid about 0.1wt % sodium benzoate. In some embodiments, the pharmaceuticalcompositions further comprise free adipic acid (free CCF that is not aCCF of the co-crystals of the invention). Such adipic acid is in aconcentration of, for example, about 5 mg/[g vehicle] to about 10 mg/[gvehicle], such as about 8.8 mg/[g vehicle].

Any orally acceptable dosage form including, but not limited to,capsules, tablets, aqueous suspensions or solutions, can be used for theoral administration. In the case of tablets for oral use, carrierscommonly used include, but are not limited to, lactose and corn starch.Lubricating agents, such as magnesium stearate, are also typicallyadded. For oral administration in a capsule form, useful diluentsinclude lactose and dried cornstarch. When aqueous suspensions arerequired for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

Microencapsulated forms with one or more excipients as noted above canalso be used in the invention. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use.

Sterile injectable forms may be aqueous or oleaginous suspension. Thesesuspensions may be formulated according to techniques known in the artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,such as carboxymethyl cellulose or similar dispersing agents which arecommonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms mayalso be used for the purposes of formulation.

In order to prolong the effect of the active compounds administered, itis often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the active compound inbiodegradable polymers such as polylactide-polyglycolide. Depending uponthe ratio of the active compound to polymer and the nature of theparticular polymer employed, the rate of compound release can becontrolled. Examples of other biodegradable polymers includepoly(orthoesters) and poly(anhydrides). Depot injectable formulationsare also prepared by entrapping the compound in liposomes ormicroemulsions that are compatible with body tissues.

When desired the above described formulations adapted to give sustainedrelease of the active ingredient may be employed.

Compositions for rectal or vaginal administration are specificallysuppositories which can be prepared by mixing the active compound withsuitable non-irritating excipients or carriers such as cocoa butter,polyethylene glycol or a suppository wax which are solid at ambienttemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the active compound.

Dosage forms for topical or transdermal administration includeointments, pastes, creams, lotions, gels, powders, solutions, sprays,inhalants or patches. The active component is admixed under sterileconditions with a pharmaceutically acceptable carrier and any neededpreservatives or buffers as may be required. Ophthalmic formulation,eardrops, and eye drops are also contemplated as being within the scopeof this invention. Additionally, transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody, can also be used. Such dosage forms can be made by dissolving ordispensing the compound in the proper medium. Absorption enhancers canalso be used to increase the flux of the compound across the skin. Therate can be controlled by either providing a rate controlling membraneor by dispersing the compound in a polymer matrix or gel.

Alternatively, the active compounds and pharmaceutically acceptablecompositions thereof may also be administered by nasal aerosol orinhalation. Such compositions are prepared according to techniqueswell-known in the art of pharmaceutical formulation and may be preparedas solutions in saline, employing benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other conventional solubilizing or dispersingagents.

The co-crystals or pharmaceutical compositions of the invention also canbe delivered by implantation (e.g., surgically) such with an implantabledevice. Examples of implantable devices include, but are not limited to,stents, delivery pumps, vascular filters, and implantable controlrelease compositions. Any implantable device can be used to deliver acompound of formula I as the active ingredient in the co-crystals orpharmaceutical compositions of this invention, provided that 1) thedevice, compound of formula I, and any pharmaceutical compositionincluding the compound are biocompatible, and 2) that the device candeliver or release an effective amount of the compound to confer atherapeutic effect on the treated patient.

Delivery of therapeutic agents via stents, delivery pumps (e g.,mini-osmotic pumps), and other implantable devices is known in the art.See, e.g., “Recent Developments in Coated Stents” by Hofma et al.,published in Current Interventional Cardiology Reports, 2001, 3: 28-36,the entire contents of which, including references cited therein, areincorporated herein. Other descriptions of implantable devices, such asstents, can be found in U.S. Pat. Nos. 6,569,195 and 6,322,847, and PCTInternational Publication Numbers WO 04/0044405, WO 04/0018228, WO03/0229390, WO 03/0228346, WO 03/0225450, WO 03/0216699, and WO03/0204168, each of which (as well as other publications cited herein)is incorporated herein in its entirety.

The active compounds and pharmaceutically acceptable compositionsthereof can be formulated in unit dosage form. The term “unit dosageform” refers to physically discrete units suitable as unitary dosage forsubjects undergoing treatment, with each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, optionally in association with a suitablepharmaceutical carrier. The unit dosage form can be for a single dailydose or one of multiple daily doses (e.g., about 1 to 4 or more timesper day). When multiple daily doses are used, the unit dosage form canbe the same or different for each dose. The amount of the activecompound in a unit dosage form will vary depending upon, for example,the host treated, and the particular mode of administration, forexample, from about 0.1 to about 200 mg/kg body weight/day.

In one embodiment, the invention is directed to methods for potentiatinga therapeutic regimen for treatment of cancer. The methods comprise thestep of administering to an individual in need thereof an effectiveamount of a co-crystal of the invention or pharmaceutical compositionthereof. The compounds of formula I and co-crystals thereof, withoutbeing bound to a particular theory, can inhibit DNA-PK. DNA-PK plays animportant role in cellular survival, for example, of cancer cells, afterDNA damage via its activity repairing double strand breaks (DSBs) bynon-homologous end joining (NHEJ). Targeting DNA-PK therefore canimprove cancer patient outcomes especially in cancer patients whoreceive therapies to induce DSBs in tumor cells since the DSBs in thetumor cells cannot be repaired and rapidly lead to cell death. In someembodiments, the methods of the invention potentiate therapeutic regimento induce DSBs. Examples of such therapies include radiation therapy(RT) and certain chemotherapies such as topoisomerase I inhibitors(e.g., topotecan, irinotecan/SN38, rubitecan and other derivatives),topoisomerase II inhibitors (e.g., etoposide and doxil), DNAintercalators (e.g., doxorubicin or epirubicin), radiomimetics (e.g.,bleomycin), PARP inhibitors (e.g., BMN-673), DNA-repair inhibitors(e.g., carboplatin), DNA cross-linkers (e.g., cisplatin), inhibitors ofthymidylate synthase (e.g., fluorouracil (5-FU)), mitotic inhibitors(e.g., paclitaxel), EGFR inhibitors (e.g., erlotinib), and EGFRmonoclonal antibodies (e.g., cetuximab).

In one specific embodiment, said potentiated therapeutic regimen fortreatment cancer includes at least one chemotherapy selected from atopoisomerase I inhibitor, topoisomerase II inhibitor, DNA intercalator,radiomimetic, PARP inhibitor, DNA-repair inhibitor, DNA cross-linkers,inhibitor of thymidylate synthase, mitotic inhibitor, EGFR inhibitor,EGFR monoclonal antibody, or radiation. In another specific embodiment,the therapeutic regimen for treatment of cancer includes radiationtherapy. The co-crystals or pharmaceutical compositions of the inventionare useful in instances where radiation therapy is indicated to enhancethe therapeutic benefit of such treatment. In addition, radiationtherapy frequently is indicated as an adjuvant to surgery in thetreatment of cancer. In general a goal of radiation therapy in theadjuvant setting is to reduce the risk of recurrence and enhancedisease-free survival when the primary tumor has been controlled. Forexample, adjuvant radiation therapy is indicated in cancers, includingbut not limited to, breast cancer, colorectal cancer, gastric-esophagealcancer, fibrosarcoma, glioblastoma, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, lung cancer, pancreatic cancer,and prostate cancer as described below. In yet another specificembodiment, the therapeutic regimen for treatment of cancer includesboth radiation therapy and a chemotherapy of at least one chemotherapyagents selected from topoisomerase I inhibitors, topoisomerase IIinhibitors, DNA intercalators, radiomimetics, PARP inhibitors,DNA-repair inhibitors, DNA cross-linkerss, inhibitors of thymidylatesynthase, mitotic inhibitors, EGFR inhibitors, or EGFR monoclonalantibodies.

In another embodiment, the invention provides methods of inhibiting orpreventing repair of DNA-damage by homologous recombination in cancerouscells. Another embodiment provides methods of promoting cell death incancerous cells. Yet another embodiment provides methods or preventingcell repair of DNA-damage in cancerous cells.

The invention further relates to sensitizing (e.g., radiosensitizing)tumor cells by utilizing a co-crystal or pharmaceutical composition ofthe invention. Accordingly, such a co-crystal or pharmaceuticalcomposition can “radiosensitize” a cell when administered to animals intherapeutically effective amount to increase the sensitivity of cells toelectromagnetic radiation and/or to promote the treatment of diseasesthat are treatable with electromagnetic radiation (e.g., X-rays).Diseases that are treatable with electromagnetic radiation includeneoplastic diseases, benign and malignant tumors, and cancerous cells.In some embodiments, the invention further relates to sensitizing tumorcells to DNA-damaging agents.

The present invention also provides methods of treating cancer in ananimal that includes administering to the animal an effective amount ofa compound of formula (I) or a co-crystal thereof, or a pharmaceuticalcomposition of the invention. The invention further is directed tomethods of inhibiting cancer cell growth, including processes ofcellular proliferation, invasiveness, and metastasis in biologicalsystems. Methods include use of such a co-crystal or pharmaceuticalcomposition to inhibit cancer cell growth. Preferably, the methods areemployed to inhibit or reduce cancer cell growth, invasiveness,metastasis, or tumor incidence in living animals, such as mammals.Methods of the invention also are readily adaptable for use in assaysystems, e.g., assaying cancer cell growth and properties thereof, aswell as identifying compounds that affect cancer cell growth.

Tumors or neoplasms include growths of tissue cells in which themultiplication of the cells is uncontrolled and progressive. Some suchgrowths are benign, but others are termed “malignant” and can lead todeath of the organism. Malignant neoplasms or “cancers” aredistinguished from benign growths in that, in addition to exhibitingaggressive cellular proliferation, they can invade surrounding tissuesand metastasize. Moreover, malignant neoplasms are characterized in thatthey show a greater loss of differentiation (greater“dedifferentiation”) and their organization relative to one another andtheir surrounding tissues. This property is also called “anaplasia.”

Neoplasms treatable by the present invention also include solid tumors,i.e., carcinomas and sarcomas. Carcinomas include those malignantneoplasms derived from epithelial cells which infiltrate (invade) thesurrounding tissues and give rise to metastases. Adenocarcinomas arecarcinomas derived from glandular tissue, or from tissues which formrecognizable glandular structures. Another broad category of cancersincludes sarcomas, which are tumors whose cells are embedded in afibrillar or homogeneous substance like embryonic connective tissue. Theinvention also enables treatment of cancers of the myeloid or lymphoidsystems, including leukemias, lymphomas, and other cancers thattypically do not present as a tumor mass, but are distributed in thevascular or lymphoreticular systems.

DNA-PK activity can be associated with various forms of cancer in, forexample, adult and pediatric oncology, growth of solidtumors/malignancies, myxoid and round cell carcinoma, locally advancedtumors, metastatic cancer, human soft tissue sarcomas, including Ewing'ssarcoma, cancer metastases, including lymphatic metastases, squamouscell carcinoma, particularly of the head and neck, esophageal squamouscell carcinoma, oral carcinoma, blood cell malignancies, includingmultiple myeloma, leukemias, including acute lymphocytic leukemia, acutenonlymphocytic leukemia, chronic lymphocytic leukemia, chronicmyelocytic leukemia, and hairy cell leukemia, effusion lymphomas (bodycavity based lymphomas), thymic lymphoma lung cancer, including smallcell carcinoma, cutaneous T cell lymphoma, Hodgkin's lymphoma,non-Hodgkin's lymphoma, cancer of the adrenal cortex, ACTH-producingtumors, non-small cell cancers, breast cancer, including small cellcarcinoma and ductal carcinoma, gastrointestinal cancers, includingstomach cancer, colon cancer, colorectal cancer, polyps associated withcolorectal neoplasia, pancreatic cancer, liver cancer, urologicalcancers, including bladder cancer, including primary superficial bladdertumors, invasive transitional cell carcinoma of the bladder, andmuscle-invasive bladder cancer, prostate cancer, malignancies of thefemale genital tract, including ovarian carcinoma, primary peritonealepithelial neoplasms, cervical carcinoma, uterine endometrial cancers,vaginal cancer, cancer of the vulva, uterine cancer and solid tumors inthe ovarian follicle, malignancies of the male genital tract, includingtesticular cancer and penile cancer, kidney cancer, including renal cellcarcinoma, brain cancer, including intrinsic brain tumors,neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cellinvasion in the central nervous system, bone cancers, including osteomasand osteosarcomas, skin cancers, including malignant melanoma, tumorprogression of human skin keratinocytes, squamous cell cancer, thyroidcancer, retinoblastoma, neuroblastoma, peritoneal effusion, malignantpleural effusion, mesothelioma, Wilms's tumors, gall bladder cancer,trophoblastic neoplasms, hemangiopericytoma, and Kaposi's sarcoma. Thus,also within the scope of this invention is a method of treating suchdiseases, which comprising administering to a subject in need thereof atherapeutically effective amount of a co-crystal of this invention or apharmaceutical composition of this invention.

In some embodiments, the invention is employed for treating lung cancer(e.g., non-small cell lung cancer (NSCLC), small cell lung cancer(SCLC), or extensive-disease small cell lung cancer (ED-SCLC)), breastcancer (e.g., triple negative breast cancer), prostate cancer, hememalignancies (e.g., acute myeloid leukemia (AML)), myeloma (e.g., plasmacell myeloma (PCM)), gastro-esophageal junction cancer (GEJ), ovariancancer, colon cancer, pharynx cancer, pancreatic cancer, gastric cancer,esophageal cancer, lymphoma (e.g., diffuse large B-cell lymphoma(DLBL)), or lung fibroblast. In some specific embodiments, the inventionis employed for treating lung cancer (e.g., non-small cell lung cancer(NSCLC), small cell lung cancer (SCLC), or extensive-disease small celllung cancer (ED-SCLC)), breast cancer (e.g., triple negative breastcancer), prostate cancer, acute myeloid leukemia, myeloma,gastro-esophageal junction cancer (GEJ), or ovarian cancer. In somespecific embodiments, the invention is employed for treating lung cancersuch as non-small cell lung cancer (NSCLC) or small cell lung cancer,such as extensive-disease small cell lung cancer (ED-SCLC). In somespecific embodiments, the invention is employed for treating breastcancer, such as triple negative breast cancer. In some specificembodiments, the invention is employed for treating gastro-esophagealjunction cancer (GEJ). In some specific embodiments, the invention isemployed for treating acute myeloid leukemia (AML).

The invention also provides a method of inhibiting DNA-PK activity in abiological sample that includes contacting the biological sample with aco-crystal or pharmaceutical composition of the invention. The term“biological sample,” as used herein, means a sample outside a livingorganism and includes, without limitation, cell cultures or extractsthereof; biopsied material obtained from a mammal or extracts thereof;and blood, saliva, urine, feces, semen, tears, or other body fluids orextracts thereof. Inhibition of kinase activity, particularly DNA-PKactivity, in a biological sample is useful for a variety of purposesknown to one of skill in the art. An example includes, but is notlimited to, the inhibition of DNA-PK in a biological assay. In oneembodiment, the method of inhibiting DNA-PK activity in a biologicalsample is limited to non-therapeutic methods.

The term “biological sample”, as used herein, includes, withoutlimitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof; blood, saliva, urine, feces,semen, tears, or other body fluids or extracts thereof.

Combination Therapies

The present invention also provides combination of chemotherapy with acompound or composition of the invention, or with a combination ofanother anticancer therapy, such as anticancer agent or radiationtherapy (or radiotherapy). In some embodiments, the compounds of formulaI and co-crystals thereof are used in combination with anotheranticancer therapy, such as anticancer drug or radiation therapy. Asused herein, the terms “in combination” or “co-administration” can beused interchangeably to refer to the use of more than one therapy (e.g.,one or more prophylactic and/or therapeutic agents). The use of theterms does not restrict the order in which therapies (e.g., prophylacticand/or therapeutic agents) are administered to a subject.

In some embodiments, said another anticancer therapy is an anti-canceragent. In other embodiments, said another anticancer therapy is aDNA-damaging agent. In yet other embodiments, said another anticancertherapy is selected from radiation therapy. In a specific embodiment,the radiation therapy is ionizing radiation.

Examples of DNA-damaging agents that may be used in combination with thecompounds of formula I and co-crystals thereof include, but are notlimited to platinating agents, such as carboplatin, nedaplatin,satraplatin and other derivatives; topoisomerase I inhibitors, such astopotecan, irinotecan/SN38, rubitecan and other derivatives;antimetabolites, such as folic family (methotrexate, pemetrexed andrelatives); purine antagonists and pyrimidine antagonists (thioguanine,fludarabine, cladribine, cytarabine, gemcitabine, 6-mercaptopurine,5-fluorouracil (5FU) and relatives); alkylating agents, such as nitrogenmustards (cyclophosphamide, melphalan, chlorambucil, mechlorethamine,ifosfamide and relatives); nitrosoureas (e.g. carmustine); triazenes(dacarbazine, temozolomide); alkyl sulphonates (eg busulfan);procarbazine and aziridines; antibiotics, such as hydroxyurea,anthracyclines (doxorubicin, daunorubicin, epirubicin and otherderivatives); anthracenediones (mitoxantrone and relatives);streptomyces family (bleomycin, mitomycin C, actinomycin); andultraviolet light.

Other therapies or anticancer agents that may be used in combinationwith the inventive agents of the present invention include surgery,radiotherapy (in but a few examples, ionizaing radiation (IR),gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy,proton therapy, brachytherapy, and systemic radioactive isotopes, toname a few), endocrine therapy, biologic response modifiers(interferons, interleukins, and tumor necrosis factor (TNF) to name afew), hyperthermia and cryotherapy, agents to attenuate any adverseeffects (e.g., antiemetics), and other approved chemotherapeutic drugs,including, but not limited to, the DNA damaging agents listed herein,spindle poisons (vinblastine, vincristine, vinorelbine, paclitaxel),podophyllotoxins (etoposide, irinotecan, vopotecan), nitrosoureas(varmustine, lomustine), inorganic ions (cisplatin, carboplatin),enzymes (vsparaginase), and hormones (tamoxifen, leuprolide, flutamide,and megestrol), Gleevec™, adriamycin, dexamethasone, andcyclophosphamide.

Additional examples of the therapeutic agents for the co-therapy of theinvention include: abarelix (Plenaxis Depot®); aldesleukin (Prokine®);Aldesleukin (Proleukin®); Alemtuzumabb (Campath®); alitretinoin(Panretin®); allopurinol (Zyloprim®); altretamine (Hexalen®); amifostine(Ethyol®); anastrozole (Arimidex®); arsenic trioxide (Trisenox®);asparaginase (Elspar®); azacitidine (Vidaza®); bevacuzimab (Avastin®);bexarotene capsules (Targretin®); bexarotene gel (Targretin®); bleomycin(Blenoxane®); bortezomib (Velcade®); busulfan intravenous (Busulfex®);busulfan oral (Myleran®); calusterone (Methosarb®); capecitabine(Xeloda®); carboplatin (Paraplatin®); carmustine (BCNU®, BiCNU®);carmustine (Gliadel®); carmustine with Polifeprosan 20 Implant (GliadelWafer®); celecoxib (Celebrex®); cetuximab (Erbitux®); chlorambucil(Leukeran®); cisplatin (Platinol®); cladribine (Leustatin®, 2-CdA®);clofarabine (Clolar®); cyclophosphamide (Cytoxan®, Neosar®);cyclophosphamide (Cytoxan Injection®); cyclophosphamide (CytoxanTablet®); cytarabine (Cytosar-U®); cytarabine liposomal (DepoCyt®);dacarbazine (DTIC-Dome®); dactinomycin, actinomycin D (Cosmegen®);Darbepoetin alfa (Aranesp®); daunorubicin liposomal (DanuoXome®);daunorubicin, daunomycin (Daunorubicin®); daunorubicin, daunomycin(Cerubidine®); Denileukin diftitox (Ontak®); dexrazoxane (Zinecard®);docetaxel (Taxotere®); doxorubicin (Adriamycin PFS®); doxorubicin(Adriamycin®, Rubex®); doxorubicin (Adriamycin PFS Injection®);doxorubicin liposomal (Doxil®); dromostanolone propionate(Dromostanolone®); dromostanolone propionate (masterone Injection®);Elliott's B Solution (Elliott's B Solution®); epirubicin (Ellence®);Epoetin alfa (Epogen®); erlotinib (Tarceva®); estramustine (EmcytR®);etoposide phosphate (Etopophos®); etoposide, VP-16 (Vepesid®);exemestane (Aromasin®); Filgrastim (Neupogen®); floxuridine(Intraarterial®) (FUDR®); fludarabine (Fludara®); fluorouracil, 5-FU(Adrucil®); fulvestrant (Faslodex®); gefitinib (Iressa®); gemcitabine(Gemzar®); gemtuzumab ozogamicin (Mylotarg®); goserelin acetate (ZoladexImplant®); goserelin acetate (Zoladex®); histrelin acetate (HistrelinImplant®); hydroxyurea (Hydrea®); Ibritumomab Tiuxetan (Zevalin®);idarubicin (Idamycin®); ifosfamide (IFEX®); imatinib mesylate(Gleevec®); interferon alfa 2a (Roferon A®); Interferon alfa-2b (IntronA®); irinotecan (Camptosar®); lenalidomide (Revlimid®); letrozole(Femara®); leucovorin (Wellcovorin, Leucovorin®); Leuprolide Acetate(Eligard®); levamisole (Ergamisol®); lomustine, CCNU (CeeBU®);meclorethamine, nitrogen mustard (Mustargen®); megestrol acetate(Megace®); melphalan, L-PAM (Alkeran®); mercaptopurine, 6-MP(Purinethol®); mesna (Mesnex®); mesna (Mesnex Tabs®); methotrexate(Methotrexate®); methoxsalen (Uvadex®); mitomycin C (Mutamycin®);mitotane (Lysodren®); mitoxantrone (Novantrone®); nandrolonephenpropionate (Durabolin-50®); nelarabine (Arranon®); Nofetumomab(Verluma®); Oprelvekin (Neumega®); oxaliplatin (Eloxatin®); paclitaxel(Paxene®); paclitaxel (Taxol®); paclitaxel protein-bound particles(Abraxane®); palifermin (Kepivance®); pamidronate (Aredia®); pegademase(Adagen (Pegademase Bovine)®); pegaspargase (Oncaspar®); Pegfilgrastim(Neulasta®); pemetrexed disodium (Alimta®); pentostatin (Nipent®);pipobroman (Vercyte®); plicamycin, mithramycin (Mithracin®); porfimersodium (Photofrin®); procarbazine (Matulane®); quinacrine (Atabrine®);Rasburicase (Elitek®); Rituximab (Rituxan®); sargramostim (Leukine®);Sargramostim (Prokine®); sorafenib (Nexavar®); streptozocin (Zanosar®);sunitinib maleate (Sutent®); talc (Sclerosol®); tamoxifen (Nolvadex®);temozolomide (Temodar®); teniposide, VM-26 (Vumon®); testolactone(Teslac®); thioguanine, 6-TG (Thioguanine®); thiotepa (Thioplex®);topotecan (Hycamtin®); toremifene (Fareston®); Tositumomab (Bexxar®);Tositumomab/I-131 tositumomab (Bexxar®); Trastuzumab (Herceptin®);tretinoin, ATRA (Vesanoid®); Uracil Mustard (Uracil Mustard Capsules®);valrubicin (Valstar®); vinblastine (Velban®); vincristine (Oncovin®);vinorelbine (Navelbine®); zoledronate (Zometa®) and vorinostat(Zolinza®).

For a comprehensive discussion of updated cancer therapies see,nci.nih.gov, a list of the FDA approved oncology drugs atfda.gov/cder/cancer/druglistframe.htm, and The Merck Manual, SeventeenthEd. 1999.

Some embodiments comprising administering to said patient an additionaltherapeutic agent selected from a DNA-damaging agent, wherein saidadditional therapeutic agent is appropriate for the disease beingtreated, and said additional therapeutic agent is administered togetherwith said compound as a single dosage form or separately from saidcompound as part of a multiple dosage form.

In some embodiments, said DNA-damaging agent is selected from at leastone from radiation, (e.g., ionizing radiation), radiomimeticneocarzinostatin, a platinating agent, a topoisomerase I inhibitor, atopoisomerase II inhibitor, an antimetabolite, an alkylating agent, analkyl sulphonates, an antimetabolite, a PARP inhibitor, or anantibiotic. In other embodiments, said DNA-damaging agent is selectedfrom at least one from ionizing radiation, a platinating agent, atopoisomerase I inhibitor, a topoisomerase II inhibitor, a PARPinhibitor, or an antibiotic.

Examples of platinating agents include cisplatin, oxaliplatin,carboplatin, nedaplatin, satraplatin and other derivatives. Otherplatinating agents include lobaplatin, and triplatin. Other platinatingagents include tetranitrate, picoplatin, satraplatin, proLindac andaroplatin.

Examples of topoisomerase I inhibitors include camptothecin, topotecan,irinotecan/SN38, rubitecan and other derivatives. Other topoisomerase Iinhibitors include belotecan.

Examples of topoisomerase II inhibitors include etoposide, daunorubicin,doxorubicin, mitoxantrone, aclarubicin, epirubicin, idarubicin,amrubicin, amsacrine, pirarubicin, valrubicin, zorubicin and teniposide.

Examples of antimetabolites include members of the folic family, purinefamily (purine antagonists), or pyrimidine family (pyrimidineantagonists). Examples of the folic family include methotrexate,pemetrexed and relatives; examples of the purine family includethioguanine, fludarabine, cladribine, 6-mercaptopurine, and relatives;examples of the pyrimidine family include cytarabine, gemcitabine,5-fluorouracil (5FU) and relatives.

Some other specific examples of antimetabolites include aminopterin,methotrexate, pemetrexed, raltitrexed, pentostatin, cladribine,clofarabine, fludarabine, thioguanine, mercaptopurine, fluorouracil,capecitabine, tegafur, carmofur, floxuridine, cytarabine, gemcitabine,azacitidine and hydroxyurea.

Examples of alkylating agents include nitrogen mustards, triazenes,alkyl sulphonates, procarbazine and aziridines. Examples of nitrogenmustards include cyclophosphamide, melphalan, chlorambucil andrelatives; examples of nitrosoureas include carmustine; examples oftriazenes include dacarbazine and temozolomide; examples of alkylsulphonates include busulfan.

Other specific examples of alkylating agents include mechlorethamine,cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, melphalan,prednimustine, bendamustine, uramustine, estramustine, carmustine,lomustine, semustine, fotemustine, nimustine, ranimustine, streptozocin,busulfan, mannosulfan, treosulfan, carboquone, thioTEPA, triaziquone,triethylenemelamine, procarbazine, dacarbazine, temozolomide,altretamine, mitobronitol, actinomycin, bleomycin, mitomycin andplicamycin.

Examples of antibiotics include mitomycin, hydroxyurea; anthracyclines,anthracenediones, streptomyces family. Examples of anthracyclinesinclude doxorubicin, daunorubicin, epirubicin and other derivatives;examples of anthracenediones include mitoxantrone and relatives;examples of streptomyces family include bleomycin, mitomycin C, andactinomycin.

Examples of PARP inhibitors include inhibitors of PARP1 and PARP2.Specific examples include olaparib (also known as AZD2281 orKU-0059436), iniparib (also known as BSI-201 or SAR240550), veliparib(also known as ABT-888), rucaparib (also known as PF-01367338),CEP-9722, INO-1001, MK-4827, E7016, BMN-673, or AZD2461. In otherembodiments, the agent that inhibits or modulates PARP1 or PARP2 isVeliparib (also known as ABT-888) or Rucaparib. In other embodiments,the agent that inhibits or modulates PARP1 or PARP2 is BMN-673.

In certain embodiments, said platinating agent is cisplatin oroxaliplatin; said topoisomerase I inhibitor is camptothecin; saidtopoisomerase II inhibitor is etoposide; and said antibiotic ismitomycin. In other embodiments, said platinating agent is selected fromcisplatin, oxaliplatin, carboplatin, nedaplatin, or satraplatin; saidtopoisomerase I inhibitor is selected from camptothecin, topotecan,irinotecan/SN38, rubitecan; said topoisomerase II inhibitor is selectedfrom etoposide; said antimetabolite is selected from a member of thefolic family, the purine family, or the pyrimidine family; saidalkylating agent is selected from nitrogen mustards, nitrosoureas,triazenes, alkyl sulfonates, procarbazine, or aziridines; and saidantibiotic is selected from hydroxyurea, anthracyclines,anthracenediones, or streptomyces family.

In some embodiments, the additional therapeutic agent is radiation(e.g., ionizing radiation). In other embodiments, the additionaltherapeutic agent is cisplatin or carboplatin. In yet other embodiments,the additional therapeutic agent is etoposide. In yet other embodiments,the additional therapeutic agent is temozolomide.

In some embodiments, the additional therapeutic agents are selected fromthose that inhibit or modulate a base excision repair protein. In aspecific embodiment, the base excision repair protein is selected fromUNG, SMUG1, MBD4, TDG, OGG1, MYH, NTH1, MPG, NEIL1, NEIL2, NEIL3 (DNAglycosylases); APE1, APEX2 (AP endonucleases); LIG1, LIG3 (DNA ligases Iand III); XRCC1 (LIG3 accessory); PNK, PNKP (polynucleotide kinase andphosphatase); PARP1, PARP2 (Poly(ADP-Ribose) Polymerases); PolB, PolG(polymerases); FEN1 (endonuclease) or Aprataxin. In another specificembodiment, the base excision repair protein is selected from PARP1,PARP2, or PolB. In yet another embodiment, the base excision repairprotein is selected from PARP1 or PARP2.

In some embodiments, the method is used on a cancer cell having defectsin the ATM signaling cascade. In some embodiments, said defect isaltered expression or activity of one or more of the following: ATM,p53, CHK2, MRE11, RAD50, NBS1, 53BP1, MDC1, H2AX, MCPH1/BRIT1, CTIP, orSMC1. In other embodiments, said defect is altered expression oractivity of one or more of the following: ATM, p53, CHK2, MRE11, RAD50,NBS1, 53BP1, MDC1 or H2AX. In another embodiment, the cell is a cancercell expressing DNA damaging oncogenes. In some embodiments, said cancercell has altered expression or activity of one or more of the following:K-Ras, N-Ras, H-Ras, Raf, Myc, Mos, E2F, Cdc25A, CDC4, CDK2, Cyclin E,Cyclin A and Rb.

According to another embodiment, the method is used on a cancer, cancercell, or cell has a defect in a protein involved in base excision repair(“base excision repair protein”). There are many methods known in theart for determining whether a tumor has a defect in base excisionrepair. For example, sequencing of either the genomic DNA or mRNAproducts of each base excision repair gene (e.g., UNG, PARP1, or LIG1)can be performed on a sample of the tumor to establish whether mutationsexpected to modulate the function or expression of the gene product arepresent (Wang et al., Cancer Research 52:4824 (1992)). In addition tothe mutational inactivation, tumor cells can modulate a DNA repair geneby hypermethylating its promoter region, leading to reduced geneexpression. This is most commonly assessed using methylation-specificpolymerase chain reaction (PCR) to quantify methylation levels on thepromoters of base excision repair genes of interest. Analysis of baseexcision repair gene promoter methylation is available commercially(e.g., sabiosciences.com/dna_methylation_product/HTML/MEAH-421A).

The expression levels of base excision repair genes can be assessed bydirectly quantifying levels of the mRNA and protein products of eachgene using standard techniques such as quantitative reversetranscriptase-coupled polymerase chain reaction (RT-PCR) andimmunhohistochemistry (IHC), respectively (Shinmura et al.,Carcinogenesis 25: 2311 (2004); Shinmura et al., Journal of Pathology225:414 (2011)).

In some embodiments, the base excision repair protein is UNG, SMUG1,MBD4, TDG, OGG1, MYH, NTH1, MPG, NEIL1, NEIL2, NEIL3 (DNA glycosylases);APE1, APEX2 (AP endonucleases); LIG1, LIG3 (DNA ligases I and III);XRCC1 (LIG3 accessory); PNK, PNKP (polynucleotide kinase andphosphatase); PARP1, PARP2 (Poly(ADP-Ribose) Polymerases); PolB, PolG(polymerases); FEN1 (endonuclease) or Aprataxin.

In some embodiments, the base excision repair protein is PARP1, PARP2,or PolB. In other embodiments, the base excision repair protein is PARP1or PARP2.

In certain embodiments, the additional therapeutic agent is selectedfrom one or more of the following: cisplatin, carboplatin, gemcitabine,etoposide, temozolomide, or ionizing radiation.

In other embodiments, the additional therapeutic agents are selectedfrom one or more of the following: gemcitabine, cisplatin orcarboplatin, and etoposide. In yet other embodiments, the additionaltherapeutic agents are selected from one or more of the following:cisplatin or carboplatin, etoposide, and ionizing radiation. In someembodiments, the cancer is lung cancer. In some embodiments, the lungcancer is non-small cell lung cancer or small cell lung cancer.

In some embodiments, the anticancer therapies for the combinationtherapy of the invention include DNA-damaging agents, such astopoisomerase inhibitors (e.g. etoposide and doxil), DNA intercalators(e.g., doxorubicin, daunorubicin, and epirubicin), radiomimetics (e.g.,bleomycin), PARP inhibitors (e.g., BMN-673), DNA-repair inhibitors(e.g., carboplatin), DNA cross-linkers (e.g., cisplatin), inhibitors ofthymidylate synthase (e.g., fluorouracil (5-FU)), mitotic inhibitors(e.g., paclitaxel), EGFR inhibitors (e.g., erlotinib), EGFR monoclonalantibodies (e.g., cetuximab), and radiation (e.g., ionizing radiation).Specific examples include etoposide, doxil, gemcitabine, paclitaxel,cisplatin, carboplatin, 5-FU, etoposide, doxorubicin, daunorubicin,epirubicin, bleomycin, BMN-673, carboplatin, erlotinib, cisplatin,carboplatin, fluorouracil cetuximab, and radiation (e.g., ionizingradiation). In some embodiments, compounds of formula I and co-crystalsthereof are used in combination with at least one anticancer drugselected from etoposide, doxil, gemcitabine, paclitaxel, cisplatin,carboplatin, 5-FU, etoposide, doxorubicin, daunorubicin, epirubicin,bleomycin, BMN-673, carboplatin, erlotinib, cisplatin, carboplatin,fluorouracil, or cetuximab, and with or without radiation. In somespecific embodiments, compounds of formula I and co-crystals thereof areused in combination with etoposide and cisplatin, with or withoutradiation (e.g., ionizing radiation). In some specific embodiments,compounds of formula I and co-crystals thereof are used in combinationwith paclitaxel and cisplatin, with or without radiation (e.g., ionizingradiation). In some specific embodiments, compounds of formula I andco-crystals thereof are used in combination with paclitaxel andcarboplatin, with or without radiation (e.g., ionizing radiation). Insome specific embodiments, compounds of formula I and co-crystalsthereof are used in combination with cisplatin and 5-Fu, with or withoutradiation (e.g., ionizing radiation).

Specific examples of cancers for the combination therapy are asdescribed above. In some embodiments, the invention is employed fortreating lung cancer (e.g., non-small cell lung cancer (NSCLC),extensive-disease small cell lung cancer (ED-SCLC)), breast cancer(e.g., triple negative breast cancer), prostate cancer, acute myeloidleukemia, myeloma, esophageal cancer (e.g., gastro-esophageal junctioncancer (GEJ)), ovarian cancer, colon cancer, pharynx cancer, pancreaticcancer, lung fibroblast, and gastric cancer. In some specificembodiments, the invention is employed for treating lung cancer (e.g.,non-small cell lung cancer (NSCLC), extensive-disease small cell lungcancer (ED-SCLC)), breast cancer (e.g., triple negative breast cancer),prostate cancer, acute myeloid leukemia, myeloma, gastro-esophagealjunction cancer (GEJ), pancreatic cancer, and ovarian cancer.

In some specific embodiments, the invention provides co-therapy of thecompounds of formula I and co-crystals thereof in combination withstandard of care (e.g., doxorubicin, etoposide, paclitaxel, and/orcarboplatin), with or without radiation (e.g., ionizing radiation), fortreating lung cancer, such as non-small cell lung cancer (NSCLC) orextensive-disease small cell lung cancer (ED-SCLC).

In some specific embodiments, the invention provides co-therapy of thecompounds of formula I and co-crystals thereof in combination withstandard of care (e.g. cisplatin, 5-FU, carboplatin, paclitaxel, and/oretoposide), with or without radiation (e.g., ionizing radiation), isemployed for treating gastro-esophageal junction cancer (GEJ).

In some specific embodiments, the invention provides co-therapy of thecompounds of formula I and co-crystals thereof in combination withstandard of care (e.g., doxorubicin and/or vincristine), with or withoutradiation (e.g., ionizing radiation), in acute myeloid leukemia orchronic lymphocytic leukemia.

In some specific embodiments, the invention provides co-therapy of thecompounds of formula I and co-crystals thereof in combination withstandard of care (e.g., doxorubicin and/or epirubicin), with or withoutradiation (e.g., ionizing radiation), in breast cancer, such as triplenegative breast cancer.

In some specific embodiments, the invention provides combination therapyof the compounds of formula I and co-crystals thereof in combinationwith radiation (or ionizing radiation); cisplatin, etoposide,paclitaxel, doxorubicin or cetuximab, with or without radiation (e.g.,ionizing radiation); cisplatin and etoposide, with or without radiation(e.g., ionizing radiation); or cisplatin and paclitaxel, with or withoutradiation (e.g., ionizing radiation), for lung cancer, such as non-smallcell lung cancer (NSCLC), small cell lung cancer, or extensive-diseasesmall cell lung cancer (ED-SCLC).

In some specific embodiments, the invention provides combination therapyof the compounds of formula I and co-crystals thereof in combinationwith radiation (e.g., ionizing radiation); cisplatin with or withoutradiation (e.g., ionizing radiation); etoposide with or withoutradiation (e.g., ionizing radiation); carboplatin with or withoutradiation (e.g., ionizing radiation); 5-FU with or without radiation(e.g., ionizing radiation); cisplatin and paclitaxel, with or withoutradiation (e.g., ionizing radiation); cisplatin and 5-FU, with orwithout radiation (e.g., ionizing radiation); or carboplatin andpaclitaxel, with or without radiation (e.g., ionizing radiation), forgastro-esophageal junction cancer (GEJ).

In some specific embodiments, the invention provides combination therapyof the compounds of formula I and co-crystals thereof in combinationwith doxorubicin or epirubicin, with or without radiation (e.g.,ionizing radiation), for breast cancer, such as triple negative breastcancer.

Another embodiment provides a method of treating breast cancer with thecompounds of formula I and co-crystals thereof in combination with aplatinating agent, with or without radiation (e.g., ionizing radiation).In some embodiments, the breast cancer is triple negative breast cancer.In other embodiments, the platinating agent is cisplatin.

In some specific embodiments, the invention provides combination therapyof the compounds of formula I and co-crystals thereof in combinationwith cetuximab, with or without radiation (e.g., ionizing radiation); orcisplatin with or without radiation (e.g., ionizing radiation), forpharynx cancer, for pharynx cancer.

In some specific embodiments, the invention provides combination therapyof the compounds of formula I and co-crystals thereof in combinationwith: cisplatin with or without radiation (e.g., ionizing radiation);etoposide with or without radiation (e.g., ionizing radiation);cisplatin and 5-FU, with or without radiation (e.g., ionizingradiation); or paclitaxel with or without radiation (e.g., ionizingradiation), for lung fibroblast.

In some specific embodiments, the invention provides combination therapyof the compounds of formula I and co-crystals thereof in combinationwith: radiation (e.g., ionizing radiation); bleomycin, doxorubicin,cisplatin, carboplatin, etoposide, paclitaxel or 5-FU, with or withoutradiation (e.g., ionizing radiation) for lung cancer, such as NSCLC,pancreatic cancer, esophageal cancer, or gastric cancer.

Another embodiment provides methods for treating pancreatic cancer byadministering a compound described herein in combination with anotherknown pancreatic cancer treatment. One aspect of the invention includesadministering a compound described herein in combination withgemcitabine.

Co-administration in the combination therapies encompassesadministration of the first and second amounts of thecompounds/therapies of the co-administration in an essentiallysimultaneous manner (such as in a single pharmaceutical composition, forexample, capsule or tablet having a fixed ratio of first and secondamounts, or in multiple, separate capsules or tablets for each) or in asequential manner in either order.

When co-administration involves the separate administration of the firstamount of a compound of the invention and a second amount of anadditional therapeutic agent/therapy, they are administered sufficientlyclose in time to have the desired therapeutic effect. The invention alsocan be practiced by including another anti-cancer chemotherapeutic agentin a therapeutic regimen for the treatment of cancer, with or withoutradiation therapy. The combination of a co-crystal or pharmaceuticalcomposition of the invention with such other agents can potentiate thechemotherapeutic protocol. For example, the inhibitor compound of theinvention can be administered with etoposide, bleomycin, doxorubicin,epirubicin, daunorubicin, or analogs thereof, agents known to cause DNAstrand breakage.

In some embodiments, the compounds of formula I and co-crystals thereofused in combination with a DNA-damaging agent (e.g., etoposide,radiation), and the compounds of formula I and co-crystals thereof areadministered after the administration of the DNA-damaging therapy.Specific examples of DNA-damaging agents are described above.

In some embodiments, the forementioned one or more additional anticanceragent or therapy is employed with Compound (1) or a pharmaceuticallyacceptable salt thereof. In some embodiments, the forementioned one ormore additional anticancer agent or therapy is employed with Compound(2) or a pharmaceutically acceptable salt thereof. In some embodiments,the forementioned one or more additional anticancer agent or therapy isemployed with the adipic acid co-crystal of Compound (1) (e.g., 2:1Compound (1) to adipic acid). In some embodiments, the forementioned oneor more additional anticancer agent or therapy is employed with theadipic co-crystal of Compound (2) (e.g., 2:1 Compound (2) to adipicacid).

In some embodiments, the forementioned one or more additional anticanceragent or therapy is employed with the pharmaceutical compositions of theinvention described above.

Described below are examples of preparing and characterizing co-crystalsof this invention, which are meant to be only illustrative and not to belimiting in any way.

Example 1: Preparation of Compounds of the Invention

As used herein, all abbreviations, symbols and conventions areconsistent with those used in the contemporary scientific literature.See, e.g., Janet S. Dodd, ed., The ACS Style Guide: A Manual for Authorsand Editors, 2nd Ed., Washington, D.C.: American Chemical Society, 1997.The following definitions describe terms and abbreviations used herein:

-   BPin pinacol boronate ester-   Brine a saturated NaCl solution in water-   DCM dichloromethane-   DIEA diisopropylethylamine-   DMA dimethylacetamide-   DME dimethoxyethane-   DMF dimethylformamide-   DMSO methylsulfoxide-   EtDuPhos    (2R,5R)-1-[2-[(2R,5R)-2,5-diethylphospholan-1-yl]phenyl]-2,5-diethylphospholane-   ESMS electrospray mass spectrometry-   Et₂O ethyl ether-   EtOAc ethyl acetate-   EtOH ethyl alcohol-   HPLC high performance liquid chromatography-   IPA isopropanol-   LC-MS liquid chromatography-mass spectrometry-   Me methyl-   MeOH methanol-   MTBE methyl t-butyl ether-   NMP N-methylpyrrolidine-   PdCl₂[P(cy)₃]₂ dichloro-bis(tricyclohexylphosphoranyl)-palladium-   Ph phenyl-   RT or rt room temperature-   TBME tert-butylmethyl ether-   tBu tertiary butyl-   THF tetrahydrofuran-   TEA triethylamine-   TMEDA tetramethylethylenediamine

Example A. Preparation of2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine-4,6-d₂(Compound 1003)

As shown in step 1-i of Scheme 1, to a solution of4,6-dichloro-2-methyl-pyrimidin-5-amine (14.04 g, 78.88 mmol) stirred inmethanol-d₄ (140.4 mL) was added formic acid-d₂ (7.77 g, 161.7 mmol) andPd black (765 mg, 7.19 mmol, wetted in methanol-d₄), followed bytriethylamine (16.36 g, 22.53 mL, 161.7 mmol). The reaction mixture wassealed in a tube and stirred at RT overnight. The mixture was thenfiltered and concentrated under reduced pressure. Et₂O (250 mL) wasadded and the mixture stirred for 1 hour at RT. The resulting solidswere filtered and washed with Et₂O (×2). The filtrate was concentratedunder reduced pressure to yield 4,6-dideutero-2-methyl-pyrimidin-5-amine(Compound 1001, 5.65 g, 65% yield) as a light yellow solid: ¹H NMR (300MHz, DMSO-d₆) δ 5.25 (s, 2H), 2.40 (s, 3H). This compound was used insubsequent steps without further purification.

As shown in step 1-ii of Scheme 1, to4,6-dideutero-2-methyl-pyrimidin-5-amine (5.35 g, 48.14 mmol) in CH₃CN(192.5 mL) was added dibromocopper (16.13 g, 3.38 mL, 72.21 mmol)followed by t-butylnitrite (8.274 g, 9.54 mL, 72.21 mmol). After 1 hour,the reaction was filtered through diatomaceous earth withdichloromethane. The filtrate was washed with water/brine (1:1), theorganic layer separated, the aqueous layer extracted withdichloromethane (2×), and the combined organic layers filtered throughdiatomaceous earth and concentrated under reduced pressure. The crudeproduct was purified by medium pressure silica gel column chromatography(0-10% EtOAc/hexanes) to yield 5-bromo-4,6-dideutero-2-methyl-pyrimidine(Compound 1002, 4.1 g, 49% yield): ¹H NMR (300 MHz, methanol-d₄) δ 2.64(s, 3H).

As shown in step 1-iii of Scheme 1, a mixture of5-bromo-4,6-dideutero-2-methyl-pyrimidine (8.5 g, 48.57 mmol),bis(pinacolato)diboron (13.57 g, 53.43 mmol), and KOAc (14.30 g, 145.7mmol) in 2-methyltetrahydrofuran (102.0 mL) was degassed by flushingwith nitrogen. To this was addeddichloro-bis(tricyclohexylphosphoranyl)-palladium (PdCl₂[P(cy)₃]₂, 1.01g, 1.364 mmol) and the reaction mixture stirred in a sealed tubeovernight at 100° C. The mixture was filtered and the filtrate stirredwith Silabond® DMT silica (SiliCycle, Inc., 0.58 mmol/g, 3.53 g) for 1hour. The mixture was filtered and concentrated under reduced pressureto yield2-methyl-4,6-dideutero-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine(Compound 1003, 13.6 g, 72% purity, the major contaminant being pinacol)as a light yellow oil: ¹H NMR (300 MHz, CDCl₃) δ 2.75 (s, 3H), 1.30 (s,12H). This compound was used in subsequent steps without furtherpurification.

Example B. Preparation of(S)-8-(1-((6-chloropyrimidin-4-yl)amino)propan-2-yl)-N-methylquinoline-4-carboxamide(Compound 1013)

As shown in step 2-i of Scheme 2, 2-bromoaniline (520 g, 3.02 mol) wasmelted at 50° C. in an oven and then added to a reaction vesselcontaining stirring acetic acid (3.12 L). Methanesulfonic acid (871.6 g,588.5 mL, 9.07 mol) was then added over 15 minutes. The reaction mixturewas heated to 60° C. and methyl vinyl ketone (377 mL, 1.5 equiv.) wasadded over 5 minutes and the reaction mixture stirred for 1 hour at 90°C. After this time another 50 mL (0.2 equiv.) of methyl vinyl ketone wasadded and the reaction mixture stirred for an additional 16 hours. Theresulting dark brown solution was cooled with an ice-water bath andpoured portion-wise into a stirring solution of 50% w/w aq. NaOH (3.894L, 73.76 mol) and ice (1 kg) also cooled with an ice-water bath.Additional ice was added as required during addition to maintain thereaction temperature below 25° C. After addition was complete thereaction mixture (pH>10) was stirred for 30 minutes whilst cooling in anice/water bath. A precipitate formed which was collected by filtration,washed with water (2 L×3), and dissolved in DCM (4 L). The organics werewashed with water (2 L) and the aqueous phase back-extracted with DCM (1L). The combined organics were dried over Na₂SO₄, filtered through a padof silica gel (about 2 L), eluted with DCM and then 3% EtOAc/DCM untilall of the product came through the plug. The volatiles of the filtratewere removed at reduced pressure and the residue was triturated withhexanes (about 500 mL). The resulting solid was collected by filtration,washed with hexanes (4×500 mL), and dried under vacuum to yield8-bromo-4-methylquinoline (Compound 1004, 363 g, 54% yield) as a lighttan solid: LC-MS=222.17 (M+H); ¹H NMR (300 MHz, CDCl₃) δ 8.91 (d, J=4.3Hz, 1H), 8.06 (d, J=7.4 Hz, 1H), 7.99 (d, J=8.4 Hz, 1H), 7.42 (t, J=7.9Hz, 1H), 7.30 (d, J=4.2 Hz, 1H), 2.73 (s, 3H).

As shown in step 2-ii of Scheme 2, selenium dioxide (764.7 g, 6.754 mol)was taken up in 3.25 L of dioxane and 500 mL of water. The stirredsolution was heated to 77° C. and 8-bromo-4-methylquinoline (compound1004, 500 g, 2.251 mol) was added in one portion. The reaction mixturewas stirred at reflux for 30 minutes and then cooled with a water bathto about 45° C., at which temperature a precipitate was observed. Thesuspension was filtered through diatomaceous earth which wassubsequently washed with the hot THF to dissolve any residual solids.The filtrate was concentrated to a minimum volume under reduced pressureand 2M NaOH (2.81 L, 5.63 mol) was added to achieve a pH of 8 to 9. Thereaction mixture was stirred at this pH for 30 minutes. A precipitateresulted which was collected by filtration and air-dried overnight toproduce 8-bromoquinoline-4-carbaldehyde (compound 1005) as an yellowishsolid: MS=236.16 (M+H); ¹H NMR (300 MHz, CDCl₃) δ 10.52 (s, 1H), 9.34(d, J=4.2 Hz, 1H), 9.05 (dd, J=8.5, 1.2 Hz, 1H), 8.18 (dd, J=7.5, 1.3Hz, 1H), 7.88 (d, J=4.2 Hz, 1H), 7.60 (dd, J=8.5, 7.5 Hz, 1H). Thismaterial was used as is in subsequent reactions.

As shown in step 2-iii of Scheme 2, to a stirred suspension of8-bromoquinoline-4-carbaldehyde (531.4 g, 2.25 mol) in THF (4.8 L) wasadded water (4.8 L) and monosodium phosphate (491.1 g, 4.05 mol). Themixture was cooled to 5° C. and, keeping the reaction temperature below15° C., sodium chlorite (534.4 g, 4.727 mol) was slowly addedportionwise as a solid over about 1 hour. After addition was completethe reaction mixture was stirred at 10° C. for 1 hour followed by theportionwise addition of 1N Na₂S₂O₃ (1.18 L) whilst keeping thetemperature below 20° C. The reaction mixture was stirred at RT followedby the removal of the THF under reduced pressure. The resulting aqueoussolution containing a precipitate was treated with sat′d NaHCO₃ (about 1L) until a pH of 3 to 4 was achieved. This mixture was stirred anadditional 15 minutes and the solid was collected by filtration, washedwith water (2×1 L), washed with tert-butyl methyl ether (2×500 mL), anddried in a convection oven at 60° C. for 48 hours. Additional dryingunder high vacuum provided 8-bromoquinoline-4-carboxylic acid (compound1006, 530.7 g, 94% yield from compound 1004) as a yellowish tan solid:LC-MS=252.34 (M+H); ¹H NMR (300 MHz, DMSO-d₆) δ 14.09 (s, 1H), 9.16 (d,J=4.4 Hz, 1H), 8.71 (dd, J=8.6, 1.2 Hz, 1H), 8.25 (dd, J=7.5, 1.2 Hz,1H), 8.03 (d, J=4.4 Hz, 1H), 7.64 (dd, J=8.6, 7.5 Hz, 1H).

As shown in step 2-iv of Scheme 2, to a suspension of8-bromoquinoline-4-carboxylic acid (compound 1006, 779.4 g, 3.092 mol)in DCM (11.7 L) was added anhydrous DMF (7.182 mL, 92.76 mmol). Thereaction mixture was cooled to 10° C. and oxalyl chloride (413 mL, 4.638mol) was added dropwise over 30 minutes. The reaction mixture wasstirred an additional 30 minutes after addition was complete,transferred to an evaporation flask, and the volatiles removed underreduced pressure. Anhydrous THF (2 L) was added and the volatiles wereonce more removed under reduced pressure in order to remove any residualoxalyl chloride. Anhydrous THF was added to the residue under anatmosphere of nitrogen and the resulting suspension of intermediate8-bromoquinoline-4-carboxylic acid chloride was stored for later use.Separately, the original reaction flask was thoroughly flushed withnitrogen gas to remove any residual oxalyl chloride and the flaskcharged with dry THF (1.16 L). After cooling to 5° C., aqueous methylamine (2.14 L of 40% w/w MeNH₂/water, 24.74 mol) was added followed bythe addition of additional THF (1.16 L). To this solution was addedportionwise over 1 hour the intermediate acid chloride suspension,keeping the reaction mixture temperature below 20° C. during addition.The evaporation vessel used to store the acid chloride was rinsed withanhydrous THF and aqueous MeNH₂ (500 mL) and this added to the reactionmixture, which was allowed to come to room temperature over 16 hours.The organic volatiles were removed under reduced pressure and theremaining mostly aqueous suspension diluted with water (1.5 L). Thesolids were collected by filtration, washed with water until thefiltrate had a pH of less than 11, washed with MTBE (2×800 mL), anddried in a convection oven at 60° C. to provide8-bromo-N-methyl-quinoline-4-carboxamide (Compound 1007, 740.4 g, 90%yield) as a light brown solid: LC-MS=265.04 (M+H); ¹H NMR (300 MHz,DMSO-d₆) δ 9.08 (d, J=4.3 Hz, 1H), 8.78 (d, J=4.7 Hz, 1H), 8.21 (dd,J=7.5, 1.2 Hz, 1H), 8.16 (dd, J=8.5, 1.3 Hz, 1H), 7.65 (d, J=4.3 Hz,1H), 7.58 (dd, J=8.5, 7.5 Hz, 1H), 2.88 (d, J=4.6 Hz, 3H).

As shown in step 2-v of Scheme 2,8-bromo-N-methyl-quinoline-4-carboxamide (Compound 1007, 722 g, 2.723mol) andtert-butyl-N-[2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)allyl]carbamate(Compound 1008, 925.4 g, 3.268 mol) were combined in a reaction flask.Na₂CO₃ (577.2 g, 5.446 mol) was added followed by the addition of water(2.17 L). The mixture was stirred for 5 minutes, 1,4-dioxane (5.78 L)was added, and the mixture was deoxygenated by bubbling in a stream ofnitrogen gas for 30 minutes. Pd(dppf) Cl₂/DCM (44.47 g, 54.46 mmol) wasadded and deoxygenation was continued as before for an additional 30minutes. The reaction mixture was stirred at reflux for 16 hours,allowed to cool to 70° C., and water (5.42 L) was added. The mixture wascooled further with an ice-water bath and stirring continued at <10° C.for 2 hours. A precipitate resulted which was collected by filtration,washed with water (3×1 L), and washed with TBME (2×1 L). The resultingprecipitate cake was split into two equal portions. Each portion wasdissolved in THF/DCM (4 L) and poured onto a plug of Florisil® (3 Lfiltration funnel with about 1.5 L of Florisil, using DCM to wet plug).The plug was subsequently washed with MeTHF until it was determined bythin layer chromatography analysis that no product remained in thefiltrate. The filtrates from both cake portions were combined andconcentrated under reduced pressure to give an orange solid. TBME (1 L)was added and the resulting suspension was filtered. The collected solidwas washed with 800 mL of TBME and dried under high vacuum overnight toprovide tert-butyl (2-(4-(methylcarbamoyl)quinolin-8-yl)allyl)carbamate(Compound 1009, 653 g, 70% yield) as an off-white solid: LC-MS=342.31(M+H); ¹H NMR (300 MHz, CDCl₃) δ 8.93 (d, J=4.3 Hz, 1H), 8.17 (dd,J=8.4, 1.6 Hz, 1H), 7.68-7.53 (m, 2H), 7.41 (d, J=4.3 Hz, 1H), 6.09 (br.s, 1H), 5.54 (s, 1H), 5.28 (s, 1H), 5.10 (br. s, 1H), 4.33 (d, J=6.0 Hz,2H), 3.11 (d, J=4.8 Hz, 3H), 1.38 (s, 9H). Additional product (34.9 g,74% total yield) was obtained by concentrating the filtrate underreduced pressure, dissolving the residue in THF, filtering the solutionthrough a plug of Florisil® as before, washing the plug with MeTHF,concentrating the filtrate under reduced pressure, adding 250 mL ofTBME, stirring for 0.5 hours, collecting the resulting precipitate byfiltration, washing the solid with EtOAc (40 mL), acetonitrile (50 mL),and drying the solid under high vacuum overnight.

As shown in step 2-vi of Scheme 2, to a stirring suspension oftert-butyl (2-(4-(methylcarbamoyl)quinolin-8-yl)allyl)carbamate(Compound 1009, 425 g, 1.245 mol) in EtOH (4.25 L) was added 5.5M HCl iniPrOH (1.132 L, 6.225 mol). The reaction mixture was stirred at reflux(76° C. internal temp) for 30 minutes and then over 90 minutes while itwas allowed to cool to 40° C. EtOAc (2.1 L) was added and the mixturewas stirred for an additional 2 hours. The solid was collected byfiltration, washed with EtOAc, and dried under high vacuum to provide8-(3-acetamidoprop-1-en-2-yl)-N-methylquinoline-4-carboxamide (Compound1010, 357.9 g, 91% yield) as a tan solid: LC-MS=242.12 (M+H); ¹H NMR(300 MHz, methanol-d₄) δ 9.07 (d, J=4.6 Hz, 1H), 8.27 (dd, J=8.5, 1.5Hz, 1H), 7.89 (dd, J=7.2, 1.5 Hz, 1H), 7.81-7.72 (m, 2H), 5.85 (s, 1H),5.75 (s, 1H), 4.05 (s, 2H), 3.04 (s, 3H).

As shown in step 2-vii of Scheme 2, under an atmosphere of nitrogen8-(3-acetamidoprop-1-en-2-yl)-N-methylquinoline-4-carboxamide (12.4 g,43.77 mmol) andcycloocta-1,5-diene/(2R,5R)-1-[2-[(2R,5R)-2,5-diethylphospholan-1-yl]phenyl]-2,5-diethylphospholane:rhodium(+1)cation-trifluoromethanesulfonate (Rh(COD)(R,R)-Et-DuPhos-OTf, 316.3 mg,0.4377 mmol) in methanol (372.0 mL) were combined and warmed to 35-40°C. until the solids were solubilized. The reaction mixture was placed ina hydrogenation apparatus, the atmosphere replaced with hydrogen, andthe mixture agitated under 100 p.s.i. of hydrogen at 50° C. for 14hours. After cooling to RT, the mixture was filtered through a bed ofFlorisil®, which was subsequently washed with MeOH (2×50 mL). Thefiltrate was concentrated under reduced pressure and any trace waterremoved via a DCM azeotrope under reduced pressure. The residue wastriturated with 20% DCM in MTBE (2×100 mL) to afford(S)-8-(1-acetamidopropan-2-yl)-N-methylquinoline-4-carboxamide (Compound1011, 11.0 g, 88% yield, 96% e.e.) as an off-white solid: ¹H-NMR (300MHz, DMSO-d₆) δ 8.97 (d, J=4.3 Hz, 1H), 8.67 (d, J=4.7 Hz, 1H), 7.97(dd, J=8.1, 1.5 Hz, 1H), 7.88 (t, J=5.6 Hz, 1H), 7.73-7.54 (m, 2H), 7.52(d, J=4.3 Hz, 1H), 4.31 (dd, J=14.3, 7.1 Hz, 1H), 3.55-3.32 (m, 3H),2.86 (d, J=4.6 Hz, 3H), 1.76 (s, 3H), 1.28 (d, J=7.0 Hz, 3H). Theenantiomeric excess (e.e.) was determined by chiral HPLC (ChiralPac IC,0.46 cm×25 cm], flow rate 1.0 mL/min for 20 min at 30° C. (20:30:50methanol/ethanol/hexanes and 0.1% diethylamine) with a retention timefor the (R)-enantiomer of 5.0 min, and for the (S)-enantiomer of 6.7min.

As shown in step 2-viii of Scheme 2,(S)-8-(1-acetamidopropan-2-yl)-N-methylquinoline-4-carboxamide (11.0 g,38.55 mmol) in 6M aqueous HCl (192.7 mL, 1.156 mol) was warmed to 60° C.After stirring for 2 days at this temperature, the reaction mixture wascooled and an additional 20 mL of 6M HCl was added. Stirring wascontinued for an additional 2 days at 70° C. The reaction mixture wascooled with an ice bath and the pH adjusted to about 11 with 6M NaOH(aq.). The aqueous mixture was extracted with 5% MeOH/DCM and thecombined organic extracts washed with water (60 mL), brine (100 mL),dried over sodium sulfate, filtered, and concentrated under reducedpressure to afford crude product as a tan solid. This solid wassuspended in EtOAc (200 mL), cooled to 3° C. with an ice bath, and 6MHCl/i-PrOH (30 mL) was added portionwise to produce a white precipitatewhich was collected by filtration. The solid was washed with EtOAc (100mL) and dried under high vacuum to provide(S)-8-(1-aminopropan-2-yl)-N-methylquinoline-4-carboxamide,dihydrochloride [Compound 1012, 7.8 g, 61% yield, 95% purity (5%compound 1011)] as a white solid. This material was used as is insubsequent reactions.

As shown in step 2-ix of Scheme 2,8-[(1S)-2-amino-1-methyl-ethyl]-N-methyl-quinoline-4-carboxamide,hydrochloride (compound 1012, 24.0 g, 72.86 mmol) was taken up in THF(230 mL) and water (40 mL) and stirred for 5 minutes. Sodium carbonate(15.44 g, 145.7 mmol) in 100 mL of water was added and the reactionmixture stirred for 10 minutes. 4,6-Dichloropyrimidine (12.18 g, 80.15mmol) was added and the reaction mixture heated at reflux at 66° C. for2 hours. The reaction mixture was cooled to RT, diluted with 200 mL ofEtOAc, the organic layer separated, and the aqueous layer extracted with100 mL EtOAc. The combined organics were washed with water (60 mL),brine (100 mL), dried over Na₂SO₄, filtered through a bed of silica gel(100 g), and concentrated under reduced pressure. The resulting crudeproduct was triturated with 20% DCM in MBTE (200 mL) then MBTE (200 mL)to produce(S)-8-(1-((6-chloropyrimidin-4-yl)amino)propan-2-yl)-N-methylquinoline-4-carboxamide(Compound 1013, 23.15 g, 88% yield) as a white solid: ¹H NMR (300 MHz,DMSO-d₆, 70° C.) δ 8.97 (d, J=4.3 Hz, 1H), 8.38 (s, 1H), 8.20 (s, 1H),8.03 (d, J—8.5 Hz, 1H), 7.71 (d, J=6.8 Hz, 1H), 7.66-7.55 (m, 1H), 7.52(d, J=4.2 Hz, 2H), 6.63 (s, 1H), 4.46 (dd, J=14.1, 7.1 Hz, 1H), 3.67 (s,2H), 2.90 (d, J=4.6 Hz, 3H), 1.40 (d, J=7.0 Hz, 3H); [α]_(D) ²⁴=44.77(c=1.14, MeOH).

Example C. Preparation of(S)—N-methyl-8-(1-((2′-methyl-[4,5′-bipyrimidin]-6-yl-4′,6′-d₂)amino)propan-2-yl)quinoline-4-carboxamide(Compound 2)

As shown in step 3-i of Scheme 3,(S)-8-(1-((6-chloropyrimidin-4-yl)amino)propan-2-yl)-N-methylquinoline-4-carboxamide(Compound 1013, 2.542 g, 7.146 mmol),2-methyl-4,6-dideutero-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine(Compound 1003, 2.204 g, 7.146 mmol, 72% by weight), Na₂CO₃ (10.72 mL of2 M (aq.), 21.44 mmol), and Silacat® DPP Pd (SiliCycle, Inc., 1.429 g,0.3573 mmol) were taken up in dioxane (30.00 mL), the solution flushedwith nitrogen gas for 5 min, and the reaction mixture stirred at 90° C.for 16 hours. The mixture was filtered through diatomaceous earth,concentrated under reduced pressure, dissolved in DMSO, and purified byreversed-phase chromatography (10-40% CH₃CN/H₂O, 0.1% TFA). The productfractions were combined and DCM and MeOH were added, followed by theaddition of 1N NaOH until a pH of greater than 7 was obtained. Theproduct solution was extracted DCM (2×) and the combined extracts driedover Na₂SO₄, filtered, and concentrated under reduced pressure to yield(S)—N-methyl-8-(1-((2′-methyl-4′,6′-dideutero-[4,5′-bipyrimidin]-6-yl)amino)propan-2-yl)quinoline-4-carboxamide(Compound 2, 181 mg, 28% yield) as an off-white solid: ¹H NMR (300 MHz,DMSO-d₆, 70° C.) δ 8.95 (d, J=4.2 Hz, 1H), 8.47 (s, 1H), 8.35 (s, 1H),8.01 (d, J=8.4 Hz, 1H), 7.74 (d, J=7.1 Hz, 1H), 7.59 (t, J=7.8 Hz, 1H),7.50 (d, J=4.3 Hz, 1H), 7.30 (s, 1H), 7.03 (s, 1H), 4.51 (h, J=7.2 Hz,1H), 3.78 (m, 2H), 2.88 (d, J=4.6 Hz, 3H), 2.68 (s, 3H), 1.41 (d, J=7.0Hz, 3H). When2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine wasused in this reaction instead of deuterated Compound 1003, Compound 1was produced: LCMS=414.40 (M+H); ¹H NMR (300 MHz, DMSO-d₆, 70° C.) δ9.14 (s, 2H), 8.95 (d, J=4.3 Hz, 1H), 8.47 (s, 1H), 8.34 (br. s, 1H),8.02 (d, J=8.4 Hz, 1H), 7.74 (d, J=7.3 Hz, 1H), 7.59 (t, J=7.8 Hz, 1H),7.50 (d, J=4.3 Hz, 1H), 7.28 (br. s, 1H), 7.04 (s, 1H), 4.52 (h, J=7.0Hz, 1H), 3.83-3.66 (m, 2H), 2.88 (d, J=4.4 Hz, 3H), 2.68 (s, 3H), 1.42(d, J=6.9 Hz, 3H).

Example 2: General Procedure for the Formation of Co-Crystals of aCompound of Formula I and a CCF Selected from Adipic Acid, Citric Acid,Fumaric Acid), Maleic Acid), Succinic Acid, or Benzoic Acid

In general, the Co-crystals of the invention can be prepared by slurrycrystallization or HME crystallization.

In one specific example, either Compound 1 or Compound 2 was weighedinto vials and mixed with a CCF at a ratio of about 1:1.2, respectively,and stirred in a suitable solvent for 2 weeks. At the end of this timeXRPD Analysis showed new crystalline patterns. Table 1 summarizes thecompound ratios and concentrations for the formation of co-crystals ofCompound 1.

TABLE 1 Weight CCF Weight Compound 1 Volume Coformer (mg) (mg) Solvent(μL) adipic acid 6.12 14.0 CH₃CN 500 succinic acid 5.45 14.9 CH₃CN 500maleic acid 5.14 15.0 EtOAc 500 furmaric acid 5.33 15.0 CH₃CN 500 citricacid 7.45 12.8 EtOAc 500 benzoic acid 5.25 14.8 water 500

Example 3: Preparation of Compounds 1 & 2/Adipic Acid Co-Crystal

A 1 liter jacketed vessel (with overhead stirring) was charged withCompound 1 (36.04 g, 0.087 mol, 1.000 equiv.), adipic acid (16.65 g,0.114 mol, 2.614 equiv.), 1-propanol (321.00 g, 5.342 mol, 122.564equiv.) and the slurry stirred at 750 rpm. A seed of the co-crystal(0.5% co-crystal seed) was added and the reaction mixture stirred at 25°C. Co-crystal formation was monitored by removing aliquots and analyzingby Raman spectroscopy. After 114 hours it was determined that co-crystalformation was complete. The slurry was filtered using a 600 mL Mediumporosity fritted funnel until the solvent level was even with the wetcake. The mother liquor was isolated, labeled and analyzed for content.The wet cake was then washed with 1-propanol (270.0 mL, 7.49 vol.). Thewet cake solids were weighed and dried in a vacuum oven at 50° C. Thefinal yield of Compound 1/adipic acid co-crystal was 21.7 g. A similarprocedure also produced a co-crystal of Compound 1 and adipic acid. HPLCanalyses indicated a stoichiometry of about 2:1 for Compound 1 orCompound 2 to adipic acid.

Alternatively, the adipic acid co-crystals of Compound (1) was alsoprepared by HME crystallization. The HME crystallizationproof-of-concept was made at the 20 g scale on a 16 mm extruder.Compound (1) freeform and neat adipic acid were extruded with high shearmixing and elevated temperatures (e.g., 144° C. or 155°) to generatecocrystal.

Certain physical properties of free base Compound (2) and its adipicco-crystal are summarized in Table 2 below.

TABLE 2 Material Properties of the Free Base and Adipic Acid Co-crystalof Compound (2) Adipic acid Adipic acid Adipic acid cocrystal cocrystalcocrystal (80% (75% Comp. Solvent Comp. 2:20% 2:25% crystallization AA)(w/w) AA) (w/w) process (80% Hot melt Hot melt Material Comp. 2:20%extrusion extrusion Assessment Free Form AA) (w/w) process process BulkDensity 0.33 g/cc 0.14 g/cc 0.43 g/cc 0.62 g/cc Tapped 0.47 g/cc 0.25g/cc 0.60 g/cc 0.70 g/cc Density

Example 4: X-Ray Powder Diffraction Characterization

The XRPD spectra for co-crystals of the invention (see FIGS. 1-7) wererecorded at room temperature in reflection mode using a Bruker D8Advance diffractometer equipped with a sealed tube Cu source and aVantec PSD detector (Bruker AXS, Madison, Wis.). The X-ray generator wasoperating at a voltage of 40 kV and a current of 40 mA. The powdersample was placed in a silicon or PMM holder. The data were recordedover the range of 4°-45° 2 theta with a step size of 0.0140° and a dwelltime of is per step. Fixed divergence slits of 0.2 mm were used.

The XRPD pattern for co-crystals Form A and Form B of the invention (seeFIGS. 14 and 15) were recorded at room temperature in transmission modeusing a PANanalytical Empyrean diffractometer equipped with a sealedtube Cu source and a PIXCel 1D detector. The X-ray generator wasoperating at a voltage of 45 kV and a current of 40 mA. The powdersample was placed in a transmission holder and held in place with Mylarthin films. The data were recorded over the range of 4°-40° 2-theta witha step size of 0.007° and a dwell time of 1549 s per step. Thediffractometer was setup with 0.020 Solar slits, fixed ½° anti-scatterslits on the incident beam and ¼° anti-scatter slits on the diffractedside. Two scans were accumulated.

FIG. 1 shows an X-ray powder diffraction (XRPD) pattern of theco-crystal formed between Compound 1 with adipic acid. The XRPD patternshows that the co-crystal is in a mixture of Forms A and B. Somespecific XRPD peaks of the spectrum are summarized below.

TABLE 3 No. Pos. [°2Th.] Rel. Int. [%] 1 6.540282 61.33 2 7.858682 60.043 11.92977 52.67 4 12.2278 23.87 5 13.03317 29.49 6 14.22935 100 718.75679 59.81 8 19.0885 36.36

FIG. 2 shows an X-ray powder diffraction pattern of the co-crystalformed between Compound 2 with adipic acid. Some specific XRPD peaks ofthe pattern are summarized below.

TABLE 4 Pos. Rel. No. [°2Th.] Int. [%] 1 6.459033 55.29 2 7.911365 51.423 11.91567 45.41 4 12.25639 24.61 5 12.98715 34.47 6 14.19256 100 718.67692 38.85 8 19.06727 28.68

FIG. 3 shows an X-ray powder diffraction pattern of the co-crystalformed between Compound 1 with citric acid. Some specific XRPD peaks ofthe pattern are summarized below.

TABLE 5 No. Pos. [°2Th.] Rel. Int. [%] 1 7.435926 50.1 2 8.291282 19.413 11.35154 21.73 4 13.26446 100 5 15.49248 47.42 6 21.55281 20.72 723.57031 30.18

FIG. 4 shows an X-ray powder diffraction pattern of the co-crystalformed between Compound 1 and fumaric acid. Some specific XRPD peaks ofthe pattern are summarized below.

TABLE 6 Pos. No. [°2Th.] Rel. Int. [%] 1 8.264997 97.26 2 10.1077 23.4 314.97012 35.06 4 16.60917 41.79 5 17.21781 100 6 25.1975 67.75 726.01104 24.39

FIG. 5 shows an X-ray powder diffraction pattern of the co-crystalformed between Compound 1 and maleic acid. Some specific XRPD peaks ofthe pattern are summarized below.

TABLE 7 Rel. Int. No. Pos. [°2Th.] [%] 1 6.205335 15.27 2 10.43158 20.843 11.28478 40.95 4 12.41363 34.13 5 13.26101 19 6 18.86924 43.52 721.08017 31.35

FIG. 6 shows an X-ray powder diffraction pattern of the co-crystalformed between Compound 1 and succinic acid. Some specific XRPD peaks ofthe pattern are summarized below.

TABLE 8 Pos. Rel. No. [°2Th.] Int. [%] 1 8.01725 26.29 2 12.33839 42.723 14.77709 37.21 4 17.31539 12.09 5 19.56132 13.66 6 20.05503 100

FIG. 7 shows an X-ray powder diffraction pattern of the co-crystalformed between Compound 1 and benzoic acid. Some specific XRPD peaks ofthe pattern are summarized below.

TABLE 9 No. Pos. [°2Th.] Rel. Int. [%] 1 8.699594 88.63 2 13.90495 68.653 15.6186 80.96 4 17.6481 100 5 18.15049 41.75 6 20.76838 39 7 24.7229367.36

Example 5: Thermogravimetric Analysis

Thermogravimetric Analyses (TGA) were conducted on a TA Instrumentsmodel Q5000 thermogravimetric analyzer. Approximately 1-4 mg of solidsample was placed in a platinum sample pan and heated in a 90 mL/minnitrogen stream at 10° C./min to 300° C. All thermograms were analyzedusing TA Instruments Universal Analysis 2000 software V4.4A.

The thermo gravimetric analysis curves for the co-crystals of Compound(1) and adipic acid and for the co-crystals of Compound (2) and adipicacid are shown in FIGS. 8 and 9, respectively. The figures show loss ofadipic acid starting at about 150° C. in both co-crystals.

Example 6: Differential Scanning Calorimetry

Differential Scanning Calorimetry (DSC) was conducted on a TAInstruments model Q2000 calorimetric analyzer. About 1-4 mg of solidsample was placed in a crimped aluminum pinhole pan and heated in a 50mL/min nitrogen stream at 10° C./min to 300° C. All data were analyzedusing TA Instruments Universal Analysis 2000 software V4.4A.

Representative differential scanning calorimetry thermograms are shownin FIG. 10 and FIG. 11 for the co-crystals of Compound (1) and adipicacid and for the co-crystals of Compound (2) and adipic acid,respectively.

Example 7: Solid State Nuclear Magnetic Resonance Spectroscopy

Solid state NMR spectra (ss-NMR) were acquired on the Bruker-Biospin 400MHz Advance III wide-bore spectrometer equipped with Bruker-Biospin 4 mmHFX probe. Approximately 70 mg of each sample was packed into fullvolume Bruker-Biospin 4 mm ZrO2 rotors. A magic angle spinning (MAS)speed of typically 12.5 kHz was applied. The temperature of the probehead was set to 275° K to minimize the effect of frictional heatingduring spinning. A relaxation delay of 30 s seconds was used for allexperiments. The CP contact time of ¹³C CPMAS experiment was set to 2ms. A CP proton pulse with linear ramp (from 50% to 100%) was employed.The Hartmann-Hahn match was optimized on external reference sample(glycine). SPINAL 64 decoupling was used with the field strength ofapproximately 100 kHz. The chemical shift was referenced againstexternal standard of adamantane with its upfield resonance set to 29.5ppm.

Following washing with solvent, ss-NMR was used to investigate theco-crystal complexes of Compound 1 or Compound 2 with adipic acid. SeeFIGS. 12 and 13, respectively. The absence of peaks characteristic offree Compound 1, Compound 2, or adipic acid indicated pure cocrystal.

Example 8: Preparation of Polymorphic Forms A and B of Adipic AcidCo-Crystals of Compounds (1) and (2)

A. Preparation of Polymorphic Form A of Adipic Acid Co-Crystal ofCompound (1)

Polymorphic Form A of adipic acid co-crystal of Compound (1) can beobtained by hot-melt crystallization of Compound (1) and adipic acid. Aspecific example of the preparation of Form A by hot melt extrusion isdescribed below.

Adipic acid was jet milled using a Fluid Energy Model 00 Jet-O-Mizerusing following settings:

Pressure Parameter [PSI] Air Supply 100 Grinding nozzle 60 Pusher nozzle80Compound (1) was screened through a #18 mesh screen. Compound (1) andjet milled adipic acid were weighed to prepare binary blends at about80, 75 and 65% weight:weight Compound (1). The initial blends wereprepared by passed through a #30 screen and subsequent mixing in aturbular mixer for 5 minutes.

The blends were extruded using a Leistritz Nano 16 twin screw extruderwith three temperature zones and equipped with a plunger feeder. Thescrew design contained conveying, pumping and 30° and 60° kneadingelements. All experiments were performed without a die installed on theextruder. Temperature, screw speed and temperature were set as listed inthe Table below. The temperature was set and controlled to the samevalue for all three heating elements. During the extrusion the torquewas monitored and the screw speed was increased when the screw was atrisk of seizing.

Parameter Setting Feed Rate 1.5 [ml/min] 3.755 Screw speed 20 to 150[rpm] Temperature [° C.] 110 130 144 155

The transmission XRPD pattern and ¹³C NMR spectrum of Form A of adipicacid co-crystal of Compound (1) are shown in FIGS. 14 and 16,respectively. Certain peaks observed in the ¹³C NMR spectrum aresummarized below.

TABLE 10 Shift ±0.1 Intensity Peak [ppm] [% of max] 1 117.1 47.6 2 96.828.2 3 95.7 26.2 4 27.6 48.1 5 14.8 32.7 6 161.6 36.5 7 154.5 33.4 851.5 24.7 9 50.2 24.3 10 25.6 99.2 11 18.5 33.7 12 179.4 54.4 13 168.455.9 14 158.3 83.5 15 147.8 46.5 16 145.7 27.9 17 143.2 44.1 18 141.843.2 19 124.6 100.0 20 31.2 31.7 21 30.1 35.2B. Preparation of Polymorphic Form A of Adipic Acid Co-Crystal ofCompound (2)

Form A of adipic acid co-crystal of Compound (2) was prepared by acetoneslurry. 322 mg of a mixture of Form A and Form B compound 2:adipic acidco-crystal prepared as described in Example 3 and 221 mg of adipic acidwere stirred in 9.8 g of acetone at 20 to 30° C. for 30 days.Approximately 50 mg of solid was isolated by filter centrifugationthrough a 0.45 μm membrane filter using a centrifugal filter device anddried in vacuum at 20 to 30° C. for approximately 2 hours. Solid stateNMR spectra were collected as described in Example 7 with the exceptionthat the sample amount was approximately 50 mg and the relaxation delaywas set to 5 s. The ¹³C NMR spectrum of Form A of adipic acid co-crystalof Compound (2) (see FIG. 17) is essentially the same as that of Form Aof adipic acid co-crystal of Compound (1). Certain peaks observed in the¹³C NMR spectrum are summarized below.

TABLE 11 Shift ±0.1 Intensity Peak [ppm] [% of max] 1 116.9 48.3 2 96.627.4 3 95.6 23.9 4 27.5 45.6 5 14.7 36.7 6 161.4 32.9 7 153.9 15.9 851.3 22.5 9 49.9 22.2 10 25.4 100.0 11 18.3 35.8 12 179.2 55.6 13 168.249.5 14 158.2 48.2 15 147.6 46.0 16 145.5 27.1 17 143.1 45.7 18 141.644.6 19 124.4 91.9 20 31.0 30.9 21 29.9 33.4C. Preparation of Polymorphic Form B of Adipic Acid Co-Crystal ofCompound (2)

Polymorphic Form B of adipic acid co-crystal of Compound (2) can beobtained by employing spray drying. A specific example is describedbelow.

A solvent mixture for spray drying was prepared by weighing out 50 g ofmethanol and 117.5 g dichloromehane into a glass bottle and shaking. 500mg of Compound (2), 176.2 mg of adipic acid and 19.3 g of the methanoldichloromethane mixture were weighed into a clear glass vial and stirreduntil all solids were dissolved. This solution was spray dried using aBuchi mini spray drier B-290 using following setting:

Parameter Setting Inlet Temp 99° C. Aspirator 100% Pump  40% Condenser−5° C. Nozzle 1 mm Atomizer 35 mm Filter Pressure −60 mbarThe isolated material completely recrystallized at room temperature toCompound (2):adipic acid co-crystal Form B over 2 months.

The XRPD pattern and ¹³C NMR spectrum of Form B of adipic acidco-crystal of Compound (2) are shown in FIGS. 15 and 18, respectively.Certain peaks observed in the ¹³C NMR spectrum are summarized below.

TABLE 12 Shift ±0.1 Intensity Peak [ppm] [% of max] 1 117.9 42.2 2 97.325.6 3 94.0 19.6 4 26.7 64.6 5 15.7 32.7 6 161.7 45.0 7 153.8 14.5 850.7 37.0 9 25.3 84.7 10 18.8 31.8 11 179.1 61.6 12 168.3 54.1 13 158.167.2 14 147.2 31.5 15 142.4 44.9 16 124.5 100.0 17 32.3 30.7 18 30.131.2 19 125.8 70.3

Example 9: Binary Phase Diagram of Compound (2) Adipic Acid Co-Crystal

FIG. 18 is a depiction of an approximate phase diagram consistent withthe measured thermal data T_(AA): Melting temperature of adipic acid,T_(CoX) melting temperature of the Compound (2): adipic acid co-crystal,T_(P): peritectic temperature, T_(CMPD2): Melting temperature ofCompound (2), T_(E1): Eutectic melt temperature, P peritectic point, E1eutectic point, S_(AA): Solid adipic acid, L liquid, S_(CoX): SolidCompound (2):Adipic Acid co-crystal, S_(CMPD2):Solid Compound (2),T_(m-E): metastable Eutectic melt temperature, m-E: metastable Eutecticpoint.

The binary phase diagram was explored using differential scanningcalorimetry on mixtures of Compound (2) and adipic acid and mixtures ofCompound (2):Adipic acid and co-crystal. The stoichiometric compositionof the co-crystal in % w:w Compound (2) was calculated from the molarstoichiometry. A representative differential scanning calorimetrythermogram is shown FIG. 11. The thermogram of Compound (2):adipic acidco-crystal shows a melting endotherm at 196° C.±2° C. followed by arecrystallization exotherm which is followed by a broad dissolutionendotherm. Melting of Compound (2) is observed at 256° C.±2° C. when theadipic acid is allowed to fully decompose and evaporate. The observeddifferential scanning calorimetry thermogram depends on the compositioni.e., the solid phases that are present in the material and is explainedby the binary phase diagram. Furthermore, it depends on otherexperimental details. A eutectic melt endotherm was observed when excessadipic acid was present in addition to the co-crystal at 138° C.±2° C.The binary phase diagram of Compound (2) and adipic acid is consistentwith the observed differential scanning calorimetry curves on compound1:adipic acid co-crystal and compound 1:adipic acid, adipic acidmixtures; an example is given in FIG. 10.

Certain measured points of the phase diagram of FIG. 19 are summarizedbelow:

TABLE 13 Composition Tempereature [% w:w] Point [° C.] ± 2 compound 2 E1T_(E1) = 138 65 ± 5 P or E2 T_(P) or T_(E2) = 196 Not known T_(AA) 153 0 T_(CMPD1) 256 100

Example 9. Biopharmaceutical Analysis

The pH solubility curve for Compound (2), Compound (2): adipic acidco-crystal, and Compound (2): adipic acid co-crystal in the presence ofexcess adipic acid were calculated from the pK_(a) values of Compound(2) and adipic acid, Compound (2):adipic acid co-crystal K_(sp) value,the binding constant of Compound (2) and adipic acid in aqueous bufferand the Compound (1) self association constant in aqueous buffer and thesolubility of Compound (2) free form. The solubility of the adipic acidcocrystal of Compound (2) was dependent on pH and the concentration ofexcess adipic acid. In general, as the concentration of adipic acidincreased the apparent solubility of the cocrystal decreased. At low pHthe solubility of the cocrystal was less than the freebase Compound (2),but within the pH range of the fasted human small intestine thecocrystal was much more soluble than the free form (or free base)Compound (2), as shown in FIG. 20. Simulations of oral dosing showed theadipic acid cocrystal drived nearly complete absorption at doses up to1.5 g, and at doses exceeding 800 mg the negative impact of adipic acidon cocrystal solubility decreased exposure slightly (data not shown).

Example 10. Dissolution Analysis

In-vitro two stage dissolution experiments using simulated intestinaland gastric fluids were used to evaluate and predict Compounds (1) and(2) and their co-crystals with adipic acid in-vivo performance. Mostcommonly drug absorption can occur in the upper intestine and highsolubility generally indicates high in-vivo bioavailability aftersimulated intestinal fluid is added in two stage dissolution experimentsfor drugs with solubility limited bioavailability. FIG. 21 shows twostage dissolution profiles for: i) Compound 1:adipic acid co-crystalprepared by hot melt extrusion and slurry crystallization; ii) HME65:35: Compound 1:adipic acid co-crystal manufactured using hot meltextrusion with 65% w:w Compound 1 and 35% w:w adipic acid; iii) HME75:25: Compound 1:adipic acid co-crystal manufactured using hot meltextrusion with 75% w:w Compound 1 and 25% w:w adipic acid; iv) HME80:20: Compound 1:adipic acid co-crystal manufactured using hot meltextrusion with 80% w:w Compound 1 and 20% w:w adipic acid; v) SC 80:20:slurry crystallized Compound 2:adipic acid co-crystal with finalCompound 2 content of 79% w:w Compound 2 and 21% w:w adipic acid; andvi) Free Form: Compound 2 free form. As shown in FIG. 21, the two stagedissolution data on Compound 1:adipic acid co-crystal and Compound2:adipic acid co-crystal showed higher Compound 1 and Compound 2concentrations than Compound 1 or Compound 2 free form, respectively.Also, the concentration of Compound 1 for Compound 1:adipic acidco-crystal prepared by hot melt extrusion from Compound 1 and adipicacid at 65% w:w and 35% w:w performed better than slurry crystallizedCompound 2:adipic acid co-crystal or Compound 1:adipic acid co-crystalprepared by hot melt extrusion from Compound 1 and adipic acid at 75%w:w and 25% w:w and Compound 1:adipic acid co-crystal prepared by hotmelt extrusion from Compound c and adipic acid at 80% w:w and 20% w:w,respectively. Without being bound to a particular theory, this ispotentially due to the microstructure that was obtained for the eutecticsolid.

Two stage dissolution experiments were performed at least in duplicate.Fasted state simulated gastric fluid (FaSSGF) was equilibrated for 30minutes under stirring to 37° C. in a 100 ml clear class vial using awater bath consisting of a temperature controlled jacketed vessel. Thecompound 1:adipic acid co-crystal and compound 2:adipic acid co-crystalwas added and the suspension was stirred at about 130 rpm and 37° C.,respectively. Aliquots (0.5 ml) were taken at 5, 15, 30, and 60 minutes.Solids were separated by filter centrifugation using centrifuge filterunits with a 0.45 μm membrane and spinning at 5000 rpm for 5 minutes onan Eppendorff Model 5418 centrifuge. The pH of the dissolution sampleswas measured after sampling at 15 and 60 minute time points. Thesupernatants of the filtered samples were 10 fold diluted of withdiluent for HPLC Analysis. At the 65 minute timepoint fasted statesimulated intestinal fluid FaSSIF equilibrated at 37° C. was added tothe suspension and the suspension was continued to stir at 130 rpm.Aliquots (0.5 ml) were taken at 75, 90, 120 and 180 minute timepoints.Solids were separated by filter centrifugation using centrifuge filterunits with a 0.45 μm membrane and spinning at 5000 rpm for 5 minutes onan Eppendorff Model 5418 centrifuge. The pH of the dissolution sampleswas measured after sampling at 75, 90 and 180 minute time points. Thesupernatants of the filtered samples were 10 fold diluted of withdiluent for HPLC Analysis. The amounts of material and simulated fluidsused are summarized below:

Material Weight [mg] Volume FaSSGF Volume FaSSIF HME 65:35 43.5, 44.5 1016 HME 75:25 40.8, 40.3 10 16 HME 80:20 38.3, 38.1 10 16 SC 80:20 31.7,32.0, 31.6 8 12 Free Form 24.7, 25.1, 26.6 8 12The concentrations of Compounds 1 and 2 were measured using followingHPLC method, respectively:

Column “Xterra Phenyl 4.6 × 50 mm, 5.0 um” Column Temperature 30° C.Flow Rate 1.5 ml/min Injector Volume 10 ul Auto-sampler Temperature 25C. Total Run Time 3.0 mins Detector Wavelength 240 nm Needle WashSolution Methanol Sampling Rate 1 per sample Data Acquisition Time 3Mobile Phase A 0.1% TFA in Water Mobile Phase B 0.1% TFA in AcetonitrileGradient 85% Mobile Phase A 15% Mobile Phase B

Typical simulated fluid preparations were used for 2 stage dissolutionexperiments: FaSSIF was prepared by adding about 1.80 g of SodiumHydroxide Pellets, 2.45 g of Maleic Anhydride, 6.37 g of SodiumChloride, 1.61 g of Sodium Taurocholate and 618.8 mg of Lecithin to 800ml water. The solution was stirred until all materials were completelydissolved. Then the pH was adjusted to 6.5 using 1.0N HCl and 50% NaOHSolution while the solution was being stirred. Water was added to afinal volume of 1 l. FaSSGF was prepared by adding 50.0 mL of 1.0N HCl,about 1.0 g of “800-2500 U/mg” pepsin, 43 mg of Sodium Taurocholate, 2.0g of Sodium Chloride (NaCl) to 800 ml water. Water was added to a finalvolume of 1 l. The final pH was typically 1-2.

Example 12. Bioavailability of the Co-Crystals of the Invention

The oral bioavailability of Compound 2:adipic acid co-crystal andCompound 2 free form in humans was predicted based on the calculated pHsolubility curves in FIG. 20 using GastroPlus, version 8.5.0002Simulations Plus, Inc. A jejunum permeability of 1.67e-4 cm/s andparticle radius of 10 microns was used. All other parameters were thedefault settings of the software. The simulations predict 100% fractionabsorbed for oral doses up to 1500 mg Compound 2:adipic acid co-crystaland Compound 2; adipic acid co-crystal with additional adipic acidpresent whereas the predicted Compound 2 oral fraction absorbed steeplydecreases with increasing doses. As shown in FIG. 22, the simulationsindicate that the compound 2:adipic acid co-crystal has superior oralbioavailability when compared to Compound 2 free form to give sufficientexposure for human safety studies for doses up to but not limited to1500 mg and can result in larger safety margins for Compound 2.Furthermore, high oral bioavailability will reduce the oral dose that isneeded to reach efficacious blood levels. Similar results are expectedfor Compound 1 based on the similarity in the observed physicalproperties of Compound 1 and Compound 2.

Example 13. Biological Efficacy of Compound 2/Adipic Acid Co-CrystalExample A. DNA-PK Kinase Inhibition Assay

The adipic acid co-crystal of Compound 2 was screened for its ability toinhibit DNA-PK kinase using a standard radiometric assay. Briefly, inthis kinase assay the transfer of the terminal ³³P-phosphate in ³³P-ATPto a peptide substrate is interrogated. The assay was carried out in384-well plates to a final volume of 50 μL per well containingapproximately 6 nM DNA-PK, 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 25 mMNaCl, 0.01% BSA, 1 mM DTT, 10 μg/mL sheared double-stranded DNA(obtained from Sigma), 0.8 mg/mL DNA-PK peptide(Glu-Pro-Pro-Leu-Ser-Gln-Glu-Ala-Phe-Ala-Asp-Leu-Trp-Lys-Lys-Lys,obtained from American Peptide), and 100 μM ATP. Accordingly, compoundsof the invention were dissolved in DMSO to make 10 mM initial stocksolutions. Serial dilutions in DMSO were then made to obtain the finalsolutions for the assay. A 0.75 μL aliquot of DMSO or inhibitor in DMSOwas added to each well, followed by the addition of ATP substratesolution containing ³³P-ATP (obtained from Perkin Elmer). The reactionwas started by the addition of DNA-PK, peptide and ds-DNA. After 45 min,the reaction was quenched with 25 μL of 5% phosphoric acid. The reactionmixture was transferred to MultiScreen HTS 384-well PH plates (obtainedfrom Millipore), allowed to bind for one hour, and washed three timeswith 1% phosphoric acid. Following the addition of 50 μL of Ultima Gold™high efficiency scintillant (obtained from Perkin Elmer), the sampleswere counted in a Packard TopCount NXT Microplate Scintillation andLuminescence Counter (Packard BioScience). The K_(i) values werecalculated using Microsoft Excel Solver macros to fit the data to thekinetic model for competitive tight-binding inhibition. The adipic acidco-crystal of Compound (2) had a Ki of about 2 nM.

Example B: Efficacy of Compounds (1) and (2) in Combination with WholeBody IR

The in vivo efficacies of Compounds (1) and (2) in combination withwhole body IR were examined in the OD26749 primary NSCLC (non-small celllung cancer) and the OE-19 GEJ cell line xenograft models. The resultsare summarized in Tables 14 and 15. In these studies, Compounds (1) and(2) were formulated with 16% captisol/1% PVP/1% HPMC E5 pH2.

B.1. Efficacy of Compound (1) in Combination with IR in the OD26749NSCLC Xenograft Model

The in vivo efficacy of Compound (1) was evaluated in the primaryOD26749 NSCLC subcutaneous xenograft model. Compound (1) administered at100 mg/kg tid on a single day significantly enhanced the radiationeffect of a single 2 Gy dose of whole body IR in this model (% T/C 26for the combination compared to % T/C of 80 for radiation alone,P<0.001). Efficacy was evaluated using a regimen in which 2-Gy wholebody IR was administered twice, one week apart. Compound (1) wasadministered PO (tid at 0, 3, and 7 h) at 100 mg/kg alone or with asingle 2 Gy dose of whole body IR at 3.25 h. Seven days later, the sameregimens were repeated. Compound (1) in combination with 2 Gy whole bodyIR induced significant tumor regression (% T/Ti of −75; P<0.01) comparedto IR alone.

Compound (1) alone and IR alone did not induce significant (P>0.05)tumor growth inhibition compared to vehicle controls (% T/C of 74 and64, respectively). In this primary tumor model, both groups exhibitedsome degree of body weight loss (6.7% and 8.7% maximal loss on Day 2 orDay 9 for the IR alone and combination group, respectively) thatrecovered over the course of the study. The addition of a secondadministration of 2 Gy IR in combination with Compound (1) resulted in asignificant increase in time to tumor doubling (TTD) with a 33.4 day TTDin the combination group compared to only 2 to 3 days for the vehicle,IR and Compound (1) single agent groups.

B.2. Bridging Study: Compounds (1) and (2) in Combination with TwoCycles of Whole Body IR in a Primary NSCLC Xenograft Model (OD26749) inNude Mice

The efficacies of Compounds (1) and (2) in combination with whole bodyIR (2 Gy) were evaluated in the OD26749 primary NSCLC xenograft model ata Compound (1) dose level of 100 mg/kg PO bid (0 and 4 h) and Compound(2) dose levels of 50 mg/kg and 100 mg/kg PO bid (0 and 4 h). Two cyclesof whole body IR (2 Gy) were given 15 min after the first compoundadministration (0.25 hour). Control animals were administered vehicle PObid (0 and 4 h). Two cycles of treatment were performed on Day 0 and Day7.

Two cycles of whole body radiation (2 Gy) alone did not inhibit tumorgrowth compared to vehicle treated tumors (% T/C=106). However, efficacywas significantly enhanced when Compounds (1) and (2) were combined withIR, as average tumor volumes in all combination groups weresignificantly smaller than those in the IR only group (P<0.001). Inaddition, the Compounds (1) and (2) (100 mg/kg bid) combination groupsdemonstrated very similar anti-tumor activity (% T/C=4.80 and 7.80respectively), blood exposure (AUC 65.8 and 58.2 μg*h/mL), andtolerability (maximum body weight change −2.40% and 2.70%). In addition,the average tumor volume in the 50-mg/kg combination group wasstatistically different than those in the Compounds (1) and (2) 100mg/kg combination groups (P<0.001).

B.3. Efficacy of Compounds (1) and (2) in Combination with IR in theOE-19 Gastro-Esophageal Junction (GEJ) Cancer Xenograft Model

The OE-19 cell line xenograft model was used to evaluate the efficacy ofCompounds (1) and (2) alone and in combination with IR. Two cycles oftreatment were administered (Day 0 and Day 7) as performed in theOD26749 model above. Two cycles of whole body IR (2 Gy) alone exhibitedminimal effect on tumor growth compared to vehicle control (% T/C=60.0)indicating that this tumor model is relatively resistant to IR. Incontrast, the combination of Compound (2) and 2 Gy whole body IRresulted in significant tumor growth inhibition compared to the vehiclecontrol with a % T/C of 8.00 (P<0.001). The combination group alsoshowed significant tumor growth inhibition compared to the IR only group(P<0.001). Compound (1) in combination with 2 Gy whole body IR alsosignificantly inhibited tumor growth in this model.

B.4. Efficacy of Compounds (1) and (2) in a Primary NSCLC XenograftModel

The in vivo efficacies of Compounds (1) and (2) were evaluated alone andin combination with three consecutive days of focused IR in the primaryLU-01-0030 NSCLC subcutaneous xenograft model. The dose-dependentanti-tumor activity of Compound (2) alone and in combination withfocused beam IR was evaluated in the LU-01-0030 model. In this model, IRtreatment alone resulted in significant tumor regression; however tumorre-growth was observed approximately 20 days after the last day oftreatment. On Day 34, Compound (2) combination groups demonstratedstatistically significant (P<0.001) anti-tumor activity when compared tothe vehicle and IR only groups, with % T/Ti values of −96.3, −67.1,−96.9, and 1.6% for the 50 and 25 mg/kg bid and 50 and 25 mg/kg qdgroups, respectively. Mice in the combination treatment groups weremonitored (without treatment) for up to 90 days as some mice had noevidence of tumor burden. In all experimental groups, treatments weregenerally well tolerated as evidenced by maximum body weight lossesranging from −1.11% to −6.93% 1 to 9 days after treatment initiation

B.5. Efficacy of Compounds (1) and (2) in Combination with IR in aPrimary GEJ Cancer Xenograft Model

The in vivo activities of Compounds (1) and (2) were compared incombination with focused beam IR in a primary gastric cancersubcutaneous xenograft model. In the ST 02 0004 model, focused IR wasadministered on three consecutive days alone and in combination withCompound (1) or Compound (2). IR treatment alone resulted in a slightdelay in tumor growth of approximately 7 days after the last day oftreatment. Compounds (1) and (2) combination groups demonstratedstatistically significant (P<0.001) anti tumor activity when compared tothe vehicle and IR only groups with a % T/Ti value of −2.8% for the 100mg/kg Compound (1) combination group and % T/C values of 9.2 and 17.4for the 100 and 25 mg/kg Compound (2) combination groups, respectively.For all experimental groups, treatment was generally well tolerated asevidenced by maximum body weight losses ranging from −8.06% to −10.0% 10to 48 days after treatment initiation

The anti-tumor activity of Compound (2) in combination with focused-beamIR and the standard of care agents, paclitaxel and carboplatin, was alsoevaluated in the ST 02 0004 model. Treatment with paclitaxel,carboplatin, and IR was administered once per week for three weeks aloneor in combination with Compound (2). Paclitaxel/carboplatin treatmentdid not impact tumor growth nor did the combination ofpaclitaxel/carboplatin and 50 mg/kg Compound (2). However, on Day 45, 25and 50 mg/kg Compound (2) in combination with paclitaxel/carboplatin andIR demonstrated a statistically significant difference (P<0.001) inanti-tumor activity when compared to the vehicle group with % T/C valuesof 2.5 and 11.1 for the 50 and 25 mg/kg Compound (2),paclitaxel/carboplatin, IR combination groups, respectively. Further,the 50 and 25 mg/kg Compound (2), paclitaxel/carboplatin, IR combinationgroups were statistically different (P<0.05) from thepaclitaxel/carboplatin, paclitaxel/carboplatin/50 mg/kg Compound (2),and paclitaxel/carboplatin/IR groups. Compound (2) blood exposures were9.3 and 27 μg*h/mL for the 25 and 50 mg/kg Compound (2) bid groups,respectively.

In Tables 14 and 15, for example, PO bid (0, 4 h) indicates Compound (2)is administered twice (bid) at time point 0 and then 4 hours after; IR(0.25 h) qdx3 indicates radiation is administered 15 minutes (0.25 h)after the administration of Compound (2) (0 h), and once a day for 3days (qdx3); q7dx2 indicates once a week for two weeks; qod indicatesevery other day twice (e.g., Day 1 and Day 3); and paclitaxel q7d x3(−0.25 h), carboplatin q7dx3 (−0.25 h) indicates administration ofpaclitaxel and carboplatin 15 minutes prior to the administration ofCompound (2), followed by additional administration of Compound (2)after 4 hours after the first administration of Compound (2). In onespecific example, “5 mg/kg paclitaxel q7dx3 (−0.25 h), 25 mg/kgcarboplatin q7dx3 (−0.25 h), 2 Gy IR qdx3 (0.25 h), PO 50 mg/kg bid (0,4 h) qdx3” indicates that 5 mg/kg of paclitaxel and 25 mg/kg ofcarboplatin are administered 15 minutes prior to the firstadministration of Compound (2); the first administration of Compound (2)is given; radiation is administered 15 minutes after the firstadministration of Compound (2); and then the second administration ofCompound (2) is provided 4 hours after the first administration ofCompound (2).

TABLE 14 Summary of In Vivo Efficacy Studies with Compound (1) TumorModel, DNA Damaging Agent Study Groups Results % T/C (Day 20) % T/Ti(Day 20) Max. body wt loss (%) OD26749 2 Gy Radiation qd × 1 80 — −6.90(Day 2) (Primary PO 100 mg/kg tid (0, 3, 7 h) qd × 1 101 — −2.40 (Day 2)NSCLC) PO 100 mg/kg tid (0, 3, 7 h) qd × 1, 2 Gy 26.0 — −9.70 (Day 2)Whole qd × 1 (3.25 h) Body IR % T/C (Day 16) % T/Ti (Day 16) Max. bodywt loss (%) OD26749 2 Gy Radiation q7d × 2 64 — −6.7 (Day 2) (Primary PO100 mg/kg tid (0, 3, 7 h) q7d × 2 74 — weight gain NSCLC) PO 100 mg/kgtid (0, 3, 7 h) q7d × 2, 2 — −75 −8.7 (Day 9) Whole Gy q7d × 2 (3.25 h)Body IR % T/C (Day 22) % T/Ti (Day 22) Max. body wt loss (%) OD26749 2Gy Radiation qd × 3 106 — −0.90 (Day 1) (Primary PO 100 mg/kg bid (0, 4h), 2 Gy (0.25 h) 4.8 — −2.40 (Day 1) NSCLC qd × 3 bridging study)*Whole Body IR % T/C (Day 29) % T/Ti (Day 29) Max. body wt loss (%)OD26749 2 Gy Radiation q7d × 2 42 — −3.50 (Day 1) (Primary PO 200 mg/kgqd, 2 Gy IR (0.25 h) 6.5 — −6.10 (Day 1) NSCLC) q7d × 2 Whole PO 100mg/kg bid (0, 4 h), 2 Gy IR — −3.1 −3.70 (Day 8) Body IR (0.25 h) q7d ×2 PO 50 mg/kg bid (0, 4 h), 2 Gy IR (0.25 h) 11.7 — −5.50 (Day 8) q7d ×2 PO 25 mg/kg bid (0, 4 h), 2 Gy IR (0.25 h) 25.6 — −7.70 (Day 8) q7d ×2 % T/C (Day 30) % T/Ti (Day 30) Max. body wt loss (%) LU-01- 2 GyRadiation qd × 3 14.8 —  −4.0 (Day 4) 0030 PO 100 mg/kg tid (0, 3, 7 h)qd × 5 79.1 — −0.63 (Day 6) (Primary PO 100 mg/kg tid (0, 3, 7 h) qd ×3, 2 Gy — −90.6 −1.58 (Day 4) NSCLC) IR (0.25 h) qd × 3 Focused PO 100mg/kg tid (0, 3, 7 h) qd × 5, 2 Gy — −91.6 −1.68 (Day 4) IR IR (0.25 h)qd × 3 PO 100 mg/kg bid (0, 4 h) qd × 3, 2 Gy — −85.6 −1.42 (Day 4) IR(0.25 h) qd × 3 % T/C (Day 27) % T/Ti (Day 27) Max. body wt loss (%)LU-01- 2 Gy Radiation qd × 3 16.1 — −7.44 (Day 3) 0030 PO 100 mg/kg qd ×3, 2 Gy (0.25 h) IR — −76.5 −3.68 (Day 2) (Primary qd × 3 NSCLC) PO 100mg/kg bid (0, 4 h) qd × 3, 2 Gy — −90.1 −2.87 (Day 3) Focused (0.25 h)IR qd × 3 IR PO 50 mg/kg bid (0, 4 h) qd × 3, 2 Gy — −87.8 −5.70 (Day 3)(0.25 h) IR qd × 3 PO 25 mg/kg bid (0, 4 h) qd × 3, 2 Gy — −80.3 −5.81(Day 2) (0.25 h) IR qd × 3 % T/C (Day 27) % T/Ti (Day 27) Max. body wtloss (%) LU-01- 2 Gy Radiation qd × 3 16.1 — −7.44 (Day 3) 0030 PO 50mg/kg bid (0, 4 h) qd × 3, 2 Gy — −76.5 −3.68 (Day 2) (Primary (0.25 h)IR qd × 3 NSCLC) PO 50 mg/kg bid (0, 4 h) qd × 2, 2 Gy — −90.1 −2.87(Day 3) Focused (0.25 h) IR qd × 3 IR PO 25 mg/kg bid (0, 4 h) qd × 3, 2Gy — −87.8 −5.70 (Day 3) (0.25 h) IR qd × 3 PO 10 mg/kg bid (0, 4 h) qd× 3, 2 Gy — −80.3 −5.81 (Day 2) (0.25 h) IR qd × 3 % T/C (Day 31) % T/Ti(Day 31) Max. body wt loss (%) LU-01- 2 Gy Radiation qd × 3 49.3  —−4.46 (Day 2) 0030 PO 10 mg.kg bid (0, 4 h) qd × 3, 2 Gy IR — −5.3 −3.33(Day 3) (Primary (0.25 h) NSCLC) PO 50 mg/kg qd × 3, 2 Gy IR (0.25 h)4.5 — −2.07 (Day 1) Focused PO 50 mg/kg bid (0, 4 h) qd × 1, 2 Gy IR 7.2— −0.59 (Day 1) IR (0.25 h) PO 50 mg/kg bid (0, 4 h) qd × 2, 2 Gy IR —−1.7 −2.11 (Day 1) (0.25 h) PO 50 mg/kg bid (0, 4 h) qd × 3, 2 Gy IR —−14.1  −0.94 (Day 3) (0.25 h) % T/C (Day 24) % T/Ti (Day 24) Max. bodywt loss (%) LU-01- 2 Gy Radiation qd × 3 26.7 — −0.40 (Day 2) 0030 PO 10mg/kg bid (0, 4 h) qd × 3, 2 Gy IR — −29.8 −1.46 (Day 4) (Primary qd × 3(0.25 h) NSCLC) PO 25 mg/kg bid (0, 4 h) qd × 3, 2 Gy IR — −75.2 −2.03(Day 4) Focused qd × 3 (0.25 h) IR PO 50 mg/kg bid (0, 4 h) qd × 3, 2 GyIR — −87.6 −1.19 (Day 4) qd × 3 (0.25 h) PO 50 mg/kg bid (0, 4 h) qod ×2, 2 Gy — −79.9 −1.59 (Day 4) IR qd × 3 (0.25 h) % T/C (Day 18) % T/Ti(Day 18) Max. body weight loss (%) OE-19 2 Gy Radiation qd7 × 2 86.0 — −1.9 (Day 1) (GEJ cell PO 100 mg/kg bid (0, 4 h) qd7 × 2 79.0 — −1.70(Day 8) line) PO 100 mg/kg bid (0, 4 h) qd7 × 2, 2 Gy 24.0 — −3.50(Day 1) Whole IR qd7 × 2 (0.25 h) Body IR % T/C (Day 34) % T/Ti (Day 34)Max. body weight loss (%) ST-02- 2 Gy Radiation qd × 3 59.6 — −8.06 (Day48) 0004 PO 100 mg/kg qd × 3 95.6 — −6.31 (Day 14) (Primary PO 100 mg/kgbid (0, 4 h) qd × 3, 2 Gy — −2.8 −10.0 (Day 10) GEJ IR (0.25 h) tumor -bridging study)* Focused IR

TABLE 15 Summary of In Vivo Efficacy Studies with Compound (2) TumorModel, DNA Damaging Agent Study Groups Results % T/C (Day 22) % T/Ti(Day 22) Max. body wt loss (%) OD26749 2 Gy Radiation qdx3 106   — −0.90(Day 1) (Primary PO 100 mg/kg bid (0, 4 h), 2 Gy (0.25 h)  7.8 — −2.70(Day 1) NSCLC-- qdx3 bridging PO 50 mg/kg bid (0, 4 h), 2 Gy (0.25 h)27.2 — −2.10 (Day 1) study) qdx3 % T/C (Day 34) % T/Ti (Day 34) Max.body wt loss (%) LU-01-0030 2 Gy Radiation qdx3 16.9 — −4.93 (Day 3)(Primary PO 50 mg/kg bid (0, 4 h) qdx3 98.3 — −1.11 (Day 9) NSCLC) PO 50mg/kg bid (0, 4 h) qdx3, 2 Gy IR — −96.3 −6.93 (Day 3) (0.25 h) qdx3 PO25 mg/kg bid (0, 4 h) qdx3, 2 Gy IR — −67.1 −6.59 (Day 3) (0.25 h) qdx3PO 50 mg/kg qdx3, 2 Gy IR (0.25 h) — −96.9 −4.66 (Day 3) qdx3 PO 25mg/kg qdx3, 2 Gy IR (0.25 h) —  −1.6 −4.62 (Day 1) qdx3 % T/C (Day 21) %T/Ti (Day 21) Max. body wt loss (%) OE-19 2 Gy Radiation q7dx2 60.0 —−0.80 (Day 1) (GEJ cell PO 100 mg/kg bid (0, 4 h) q7dx2, 2 Gy  8.0 —−6.50 (Day 7) line-- IR q7dx2 (0.25 h) bridging study) % T/C (Day 34) %T/Ti (Day 34) Max. body wt loss (%) ST-02-0004 2 Gy Radiation qdx3 56.9—  −8.06 (Day 48) (Primary GEJ PO 100 mg/kg bid (0, 4 h) qdx3 67.6 — −7.61 (Day 34) tumor) PO 100 mg/kg bid (0, 4 h) qdx3, 2 Gy IR  9.2 — −9.15 (Day 14) qdx3 (0.25 h) PO 25 mg/kg bid (0, 4 h) qdx3, 2 Gy IR17.4 —  −6.73 (Day 48) qdx3 (0.25 h) % T/C (Day 45) % T/Ti (Day 45) Max.body wt loss (%) ST-02-0004 5 mg/kg paclitaxel q7dx3 (0 h), 25 mg/kg98.0 —  −8.93 (Day 45) (Primary GEJ carboplatin q7dx3 (0 h) tumor- 5mg/kg paclitaxel q7dx3 (−0.25 h), 25 mg/kg 95.4 —  −10.1 (Day 45) withSOC) carboplatin q7dx3 (−0.25 h), PO 50 mg/kg bid (0, 4 h) qdx3 5 mg/kgpaclitaxel q7dx3, 25 mg/kg (−0.25 h), 34.9 — −10.0 (Day 3) carboplatinq7dx3 (−0.25 h), 2 Gy IR qdx3 (0 h) 5 mg/kg paclitaxel q7dx3 (−0.25 h),25 mg/kg  2.5 —  −9.20 (Day 10) carboplatin q7dx3 (−0.25 h), 2 Gy IRqdx3 (0.25 h), PO 50 mg/kg bid (0, 4 h) qdx3 5 mg/kg paclitaxel q7dx3(−0.25 h), 25 mg/kg 11.1 — −8.21 (Day 3) carboplatin q7dx3 (−0.25 h), 2Gy IR qdx3 (0.25 h), PO 25 mg/kg bid (0, 4 h) qdx3

Example 11. The Combination of Compound (1) or Compound (2) withStandard of Care Drugs or Radiation in Cancer Cell Lines

The cell-based experiments and assays were performed with eithermolecule but not always with both. Compounds (1) and (2) were generallyvery similar in those assays and experiments. Analysis of thecombination experiments was performed using two methods: the BlissAdditivity model and the Mixtures Blend method to determine the degreeof synergy, additivity, or antagonism. In the Bliss method, a matrix ofBliss scores was generated for each cell line and treatment, and a sumof the Bliss values over the range of combination concentrations testedwas calculated. The average Bliss score (sum of Bliss divided by thenumber of total data points) was then used to categorize the cell lineand treatment as follows: greater than 10 indicates strong synergy,greater than 5 indicates synergy, between 5 and −5 indicates additivity,less than −5 indicates antagonism, and less than −10 indicates strongantagonism. Larger average Bliss values indicate greater confidence inreporting synergy, and smaller scores indicate greater confidence inreporting antagonism. In the Mixtures Blend method combinants were addedin a range of optimal ratios using design of experiment (DOE) software(DX-8 from STAT-EASE); the cells were irradiated with 2 Gy as required.Synergy was determined using statistical analysis of the data (ANOVA) toindicate linear (additivity) or statistically significant (p<0.1)non-linear (antagonism or synergy) mixes of the combinants.

Certain cancer cell lines and their tumor types are listed in Table 16.

TABLE 16 Cancer cell line list CELL LINE TUMOR TYPE DOHH-2 Lymphoma- Bcell DU-4475 Breast EOL-1 Leukemia Farage Lymphoma- non-hodgkins B cellGRANTA-519 Lymphoma- mantle cell HBL-1 Lymphoma-B cell HCC2935Lung-NSCLC HCC95 Lung-NSCLC HH Lymphoma-T cell HT-115 Colorectal JHH-2Liver KARPAS-299 Lymphoma- non-hodgkins B cell KARPAS-422 Lymphoma-non-hodgkins B cell KARPAS-620 Multiple Myeloma KASUMI-1 Leukemia, AMLKE-97 Gastric KELLY Neuroblastoma KG-1 Leukemia, AML KG-1a Leukemia, AMLKMS-20 Multiple Myeloma KMS-21-BM Multiple Myeloma KMS-34 MultipleMyeloma LC-1F Lung-NSCLC LCLC-103H Lung-NSCLC LU-134-A Lung--SCLC LU-139Lung--SCLC MDST8 Colorectal ML-1 Thyroid MOLM-13 Leukemia-CML MV-4-11Leukemia NCI-H1048 Lung--SCLC NCI-H1650 Lung-NSCLC NCI-H1694 Lung--SCLCNCI-H1944 Lung-NSCLC NCI-H1993 Lung-NSCLC NCI-H2126 Lung-NSCLC NCI-H2141Lung--SCLC NCI-H2171 Lung--SCLC NCI-H2228 Lung-NSCLC NCI-H446 Lung--SCLCNCI-H820 Lung-NSCLC NCI-H841 Lung--SCLC NCI-H929 Multiple Myeloma NOMO-1Leukemia, AML OCI-Ly3 Lymphoma- B cell OCI-Ly7 Lymphoma- B cell OPM-2Lymphoma- B cell OVK18 Lymphoma- B cell PC-3 Prostate PC-9 Lung-NSCLC RLLymphoma- B cell RPMI-8226 Lymphoma- B cell SU-DHL-10-epst Lymphoma- Bcell TE-1 Esophageal TE-14 Esophageal THP-1 Leukemia, AML U-2932Lymphoma- B cell WM-266-4 Skin WSU-NHL Lymphoma- B cell ZR-75-1 Breast

A. Double Combinations

Compound (2) was tested against a panel of 60 cancer cell lines (seeTable 16) alone and in combination with a panel of cytotoxic andnon-cytotoxic SOC agents. The 60 cancer cell lines represent linesderived from breast cancer, prostate cancer, lung cancer, acute myeloidleukemia (AML), myeloma and other cancers. Cells were removed fromliquid nitrogen storage, thawed and expanded in appropriate growthmedia. Once expanded, cells were seeded in 384-well tissue culturetreated plates at 500 cells per well. After 24 hours, cells were treatedfor either 0 hours or treated for 144 hours with Compound (2) incombination with genotoxin: bleomycin (radio mimetic), doxorubicin(topoisomerase II inhibitor), etoposide (topoisomerase II inhibitor),carboplatin (DNA crosslinker), BMN-673 (PARP inhibitor), and tarceva(EGFR inhibitor)). At the end of either 0 hours or 144 hours, cellstatus was analyzed using ATPLite (Perkin Elmer) to assess thebiological response of cells to drug combinations.

Compound (2) demonstrated strong synergy with several agents tested:etoposide (topoisomerase inhibitor), doxorubicin (DNA intercalator), andbleomycin (radiomimetic) (FIG. 23). Some synergy was seen in combinationwith BMN-673 (PARP inhibitor) and carboplatin DNA-repair inhibitor).Additivity was seen with erlotinib (EGFR inhibitor) (FIG. 23). Whenanalyzed by cancer cell line type, Compound (2) and BMN-673 demonstratedgreatest activity against AML. Compound (2) and etoposide, while highlyactive against most lines, was particularly active against non-smallcell lung cancer lines as was Compound (2) and doxorubicin (see below).Bliss synergy data of Compound (2) in various tumor types (acute myeloidleukemia (AML), diffuse large B-cell lymphoma (DLBCL), non-small celllung cancer (NSCLC), plasma cell myeloma (PCM), small cell lung cancer(SCLC)) are shown in FIGS. 24-30: combination of Compound (2) withBMN-673 in FIG. 24; combination of Compound (2) with etoposide in FIG.25; combination of Compound (2) with bleomycin in FIG. 26; combinationof Compound (2) with erlotinib in FIG. 27; combination of Compound (2)with doxorubicin in FIG. 28; combination of Compound (2) with bleomycinin FIG. 29; combination of Compound (2) with carboplatin in FIG. 30.

Combinations of Compound (2) and doxorubicin or epirubicin (DNAintercalator) were tested against breast cancer cell lines (Tables 17and 18), with a comparison between wild-type and mutant lines being afocus of the study. Independent of plating density, sensitivity todoxorubicin alone, or BRCA status, the combination of doxorubicin andCompound (2) was strongly synergistic in all five cell lines and at bothCompound (2) concentrations tested (Bliss analysis). The >3-fold shiftin IC50 of the combination of doxorubicin and Compound (2) compared todoxorubicin alone also indicates a high degree of synergy. A similarexperiment using Doxorubicin or epirubicin in combination with Compound(2) in the DU4475 breast cancer line demonstrated strong synergy (Blissanalysis) (Table 18).

The combination of the Compound (2) and doxorubicin or epirubicin wasstrongly synergistic in all triple-negative breast cancer cell linesevaluated, independent of BRCA status or plating density.

TABLE 17 Summary of Combinations with Compound (2) and Doxorubicin inTriple Negative Breast Cancer Cell Lines Plating Average DoxorubicinIC50 Maximum IC50 Cell Line Density BRCA status Bliss score (μM) Shift(fold) HCC-1395 5000 Mutant 11.1 0.5 4.6 HCC-1599 Unknown Mutant 14.30.2 4.3 HCC-1937 5000 Mutant 16.6 0.2 3.7 HCC-1937 20000 Mutant 15.6 0.63.9 MDA-MB-436 5000 Mutant 14.5 0.7 9.1 MDA-MB-436 20000 Mutant 14.4 0.34.7 MDA-MB-468 5000 Wild-type 23.1 0.02 19 MDA-MB-468 20000 Wild-type24.7 0.04 13

TABLE 18 Summary of Combinations with Compound (2) and Doxorubicin orEpirubicin in DU4475 Cells Drug Average Bliss score Doxorubicin 31.9Epirubicin 33.3

B. Double and Triple Combinations with and without Radiation (2 Gy)

The following SOC agents were tested in double combinations withCompound (1): etoposide (a topoisomerase inhibitor that induces DSBs),cisplatin (DNA cross-linker), carboplatin (DNA cross-linker),fluorouracil (5-FU, antimetabolite that inhibits thymidylate synthase),paclitaxel (mitotic inhibitor that binds to tubulin), cetuximab (EGFRmonoclonal antibody), and radiation. Other than the combination ofradiation and Compound (1), the strongest interaction for the doublecombination studies was etoposide and Compound (1) in A549 cells (Table19) and Compound (1) and etoposide in ESO26 (Table 20). These findingswere confirmed using the Bliss Additivity model (Table 21). Othercombinations demonstrated additivity with rare examples of antagonism.While there is not total agreement in the various lines tested, (asdetailed in the other sections) the majority agree and the conclusionsarrived at are common to each. The same experiment performed using OE19cells showed a more complex interaction pattern in the absence ofradiation. A significantly enhanced effect was seen when radiation wasadded to the combinations, reinforcing the strong relationship betweenDNA damage (DSB and SSB) and DNA-PK inhibition. The cancer cell lines inTable 19 indicate: ESO26—gastroesophageal junction cancer,OE19—gastroesophageal junction cancer, DMS-53—SCLC, A549—lung cancer,colo205—colon cancer, H460—lung cancer, H2009—lung cancer, FaDu—pharynxcancer, Miapaca2—pancreatic cancer, HFL1—human fetal lung fibroblast.

The combination of the Compound (2) and doxorubicin or epirubicin wasstrongly synergistic in all triple-negative breast cancer cell linesevaluated, independent of BRCA status or plating density.

In the triple SOC combination experiments, synergy was demonstrated withthe combination of etoposide, cisplatin, and Compound (1) in the DMS-53and A549 cell lines. The major driver for this synergy was thecombination of etoposide with Compound (1). Paclitaxel, cisplatin, andCompound (1) was additive in the A549 cell line, while cisplatin, 5-FUand Compound (1) were synergistic in the Colo205 cell line. A highlysignificant reduction in cell viability was observed upon the additionof radiation to these combinations, principally driven by thecontribution of Compound (1). Compound (2) demonstrated the samecombination outcomes on cell viability with SOC combinants (using asmaller set of cancer cell lines) when compared to Compound (1).

TABLE 19 Effect of Compound (1) in Combination with Genotoxic Agents onthe Viability of Cancer Cell Lines Synergy or Antagonism Combinationwith Compound (1) Plus Cis- Eto- Carbo- Pacli- 5- Cetu- No RadiationCell Line platin poside platin taxel FU ximab Radiation (2 Gy) A549 ✓Additivity ND ✓ Strong Synergy ND ✓ Antagonism ND Synergy Plus ✓Additivity Additivity, Plus ✓ ✓ Strong Synergy Strong Synergy, Plus ✓ ✓Additivity Synergy, Plus H460 Synergy Plus H2009 Additivity Plus Colo205✓ ✓ Synergy Synergy, Plus DMS-53 ✓ ✓ Strong Synergy N/A OE19 ✓ ✓ MixSynergy, Plus ✓ Antagonism Synergy*, Plus ✓ Additivity Synergy*, PlusFaDu ✓ Additivity Additivity, Plus Synergy Plus HFL1 ✓ Additive ND ✓Synergy ND ✓ ✓ Additivity Additivity, Plus ✓ Additivity ND Additivity Noeffect ND = Not Determined, N/A = Not Applicable: etoposide is aradiomimetic., Plus = enhanced effect of radiation. *Viability reductiondriven predominantly by Compound (1) plus radiation.

TABLE 20 Effect of Compound (2) in Combination with Genotoxic Agents onthe Viability of the ESO26 (GEJ) Cancer Cell Line (Mixtures analysis) 1.Combinations with Comp 2 2. No Radiation 3. Plus Radiation Cisplatin,5-FU Synergy; Comp. 2 Significant reduction in cell with 5-FU survivaldriven by Comp. 2 and radiation Carboplatin, Additive overallSignificant reduction in cell Paclitaxel survival driven by Comp. 2 andradiation Etoposide Significant synergy Not applicable Based on IC₅₀ of20 μM for Comp. 2, 50 μM for carboplatin, 1.5 μM for cisplatin, 3 nM forpaclitaxel, 0.6 μM for etoposide and 20 μM for 5-FU. Not applicable:etoposide is a radiomimetic.

TABLE 21 Effect of Compound (2) in Combination with Etoposide on theViability of Cancer Cell Lines (Bliss analysis) Cell Line Average BlissScore A549   27.3 (n = 1) ESO26 43.2 ± 6.5 (n = 3)  HFL1 8.7 ± 5.6 (n =3)

C. Effect of the Combination of Compound (1) or (2) and SOC in PrimaryTumor Chemosensitivity Assays (TCA)

Primary human tumors tested in vitro may provide a better indicator ofefficacy of DNA PK inhibition than immortalized cancer cell lines due totheir increased heterogeneity and closer proximity to the patient tumorfrom which they were derived. A panel of primary human tumors (NSCLC,pancreatic, esophageal, gastric, etc.) was treated with Compound (1) todetermine the effectiveness of DNA-PK inhibition in combination withradiation, bleomycin (a radiomimetic agent that induces DSBs),doxorubicin (DNA intercalator), cisplatin, carboplatin, etoposide,paclitaxel, or 5-FU.

Compound (1) (10× and 30×IC50) was administered in combination with adose range of bleomycin or radiation. Dissociated cells frommouse-passaged tumors were cultured for 6 days after combinationexposure and then assessed for viability using the Cell Titer-Glo assay.The Bliss Additivity statistical model was used to determine the degreeof synergy, additivity, or antagonism of each combination treatment.

The combination of Compound (1) and bleomycin or radiation was additiveor synergistic in all tumors tested (29/29) (see FIG. 31). In addition,strong synergy was seen in nearly a third of both bleomycin (9/29) andradiation (3/8) treated tumors in combination with Compound (1).Similarly, Compound (2) was tested in combination with a dose range ofbleomycin in a smaller subset of tumors (gastric, pancreatic). Thecombination of Compound (2) and bleomycin was additive or synergistic inall tumors tested (20/20) and strongly synergistic in a subset of those(3/20) (see FIG. 31). These data suggest that a DNA-PK inhibitor incombination with radiation therapy may be more broadly effective thanthe standard of care alone.

A panel of primary tumors was also treated with Compound (1) incombination with a variety of chemotherapeutic agents (gemcitabine,paclitaxel, cisplatin, carboplatin, 5-FU, etoposide) commonly used inthe treatment of the tumor types tested. Additivity was observed in mosttumors treated with Compound (1) and either gemcitabine, (2/4)paclitaxel (1/5), 5-FU (5/5), or doxorubicin (1/1). However, antagonismwas seen in some tumors with gemcitabine (2/4) and paclitaxel (1/5).Synergy or additivity was observed in nearly all tumors with bothcarboplatin (5/5) and cisplatin (9/10), but one tumor showed antagonismwith cisplatin. The combination of Compound (1) and etoposide showedstrong synergy in all tumors tested (4/4). These TCA results wereconsistent with the combination data generated using cancer cell lines.Overall, these data suggest that a selective DNA-PK inhibitor mayprovide added benefit to cancer patients receiving standard of caretreatment in a variety of clinical applications.

Example 14. Effect of Compound (1) on Clonogenic Survival of IrradiatedCancer Cell Lines

The clonogenic cell survival assay measures the ability of a cell toproliferate indefinitely, thereby retaining its self-renewing ability toform a colony (i.e., clone). This assay has been a mainstay in radiationoncology for decades and was used to determine the effect of Compound(1) on the clonogenicity of a panel of cell lines across multiple tumortypes following radiation. Compound (1) in combination with radiationwas shown to be very efficacious in decreasing the clonogenicity of allcancer cell lines tested with dose enhancement factors (DEF, thedifference in colony number at surviving fraction 0.1) ranging from 2.5to >5. Miapaca2 cells exhibited the lowest DEF (2.5), while in FaDucells, the combination of Compound (1) and radiation completelyeliminated colony formation with as little as 0.5 Gy and showed a DEFof >8. A DEF greater than 1.5 is generally considered to be clinicallymeaningful; therefore, by these standards, Compound (1) would becharacterized as a strong radio-enhancing agent. These data areconsistent with the previous cell viability data in suggesting that abroad responder population can be expected in cancer patients treatedwith Compound (1) in combination with radiation.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

All references provided herein are incorporated herein in its entiretyby reference. As used herein, all abbreviations, symbols and conventionsare consistent with those used in the contemporary scientificliterature. See, e.g., Janet S. Dodd, ed., The ACS Style Guide: A Manualfor Authors and Editors, 2nd Ed., Washington, D.C.: American ChemicalSociety, 1997.

What is claimed is:
 1. A method of potentiating a therapeutic regimenfor the treatment of breast cancer, colorectal cancer,gastric-esophageal cancer, fibrosarcoma, glioblastoma, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, lung cancer,pancreatic cancer or prostate cancer in a patient comprisingadministering to said patient an effective amount of a co-crystalcomprising a compound of the formula

and a co-crystal former, wherein the co-crystal former is adipic acid,wherein each of R¹ and R² is independently hydrogen or deuterium.
 2. Themethod of claim 1, wherein a molar ratio of the adipic acid to thecompound of formula I is about 1 to
 2. 3. The method of claim 2, whereinthe compound of formula I is(S)—N-methyl-8-(1-((2′-methyl-[4,5′-bipyrimidin]-6-yl)amino)propan-2-yl)quinoline-4-carboxamide.4. The method of claim 2, wherein the compound of formula I is(S)—N-methyl-8-(1-((2′-methyl-4′,6′-dideutero-[4,5′-bipyrimidin]-6-yl)amino)propan-2-yl)quinoline-4-carboxamide.5. The method of claim 3, wherein the compound has X-ray powderdiffraction peaks at about 6.46, 7.91, 11.92, 12.26, 12.99, 14.19,18.68, and 19.07° 2-Theta.
 6. The method of claim 3, wherein thecompound has a DSC peak in its DSC thermogram at about 195° C. and about245° C.
 7. The method of claim 4, wherein the compound has a DSC peak inits DSC thermogram at about 195° C. and about 245° C.
 8. The method ofclaim 4, wherein the compound has X-ray powder diffraction peaks atabout 6.46, 7.91, 11.92, 12.26, 12.99, 14.19, 18.68, and 19.07° 2-Theta.9. The method of claim 1, wherein the compound is administered in apharmaceutical composition.
 10. The method of claim 1, wherein thetherapeutic regimen comprises radiation therapy or chemotherapy, or bothradiation therapy and chemotherapy.
 11. The method of claim 1, whereinthe co-crystal is administered with etoposide, doxorubicin,daunorubicin, epirubicin or bleomycin.
 12. A method of treating breastcancer, colorectal cancer, gastric-esophageal cancer, fibrosarcoma,glioblastoma, hepatocellular carcinoma, head and neck squamous cellcarcinoma, melanoma, lung cancer, pancreatic cancer or prostate cancerin a patient comprising administering to said patient an effectiveamount of a co-crystal comprising a compound of the formula

and a co-crystal former, wherein the co-crystal former is adipic acid,wherein each of R¹ and R² is independently hydrogen or deuterium. 13.The method of claim 12, wherein a molar ratio of the adipic acid to thecompound of formula I is about 1 to
 2. 14. The method of claim 13,wherein the compound of formula I is(S)—N-methyl-8-(1-((2′-methyl-[4,5′-bipyrimidin]-6-yl)amino)propan-2-yl)quinoline-4-carboxamide.15. The method of claim 13, wherein the compound of formula I is(S)—N-methyl-8-(1-((2′-methyl-4′,6′-dideutero-[4,5′-bipyrimidin]-6-yl)amino)propan-2-yl)quinoline-4-carboxamide.16. The method of claim 14, wherein the compound has X-ray powderdiffraction peaks at about 6.46, 7.91, 11.92, 12.26, 12.99, 14.19,18.68, and 19.07° 2-Theta.
 17. The method of claim 14, wherein thecompound has a DSC peak in its DSC thermogram at about 195° C. and about245° C.
 18. The method of claim 15, wherein the compound has a DSC peakin its DSC thermogram at about 195° C. and about 245° C.
 19. The methodof claim 15, wherein the compound has X-ray powder diffraction peaks atabout 6.46, 7.91, 11.92, 12.26, 12.99, 14.19, 18.68, and 19.07° 2-Theta.20. The method of claim 12, wherein the compound is administered in apharmaceutical composition.
 21. The method of claim 12, wherein theco-crystal is administered with an additional therapeutic agent, whereinthe additional therapeutic agent comprises radiation therapy orchemotherapy, or both radiation therapy and chemotherapy.
 22. The methodof claim 12, wherein the co-crystal is administered with etoposide,doxorubicin, daunorubicin, epirubicin or bleomycin.